Respiratory System

Monographs on Pathology of Laboratory Animals

Sponsored by the International Life Sciences Institute

Editorial Board

J. D. Burek, West Point· J. S. Campbell, Ottawa C. C. Capen, Columbus . A. Cardesa, Barcelona RG.Carison, Kalamazoo· D. de Paola, Rio de Janeiro G. Della Porta, Milan . J. L. Emerson, Atlanta F. M. Garner, Rockville . L. Golberg, Research Triangle Park H. C. Grice, N epean . C. C. Harris, Bethesda . R Hess, Basel C. F. Hollander, Rijswijk· G. H. Hottendorf, Syracuse RD. Hunt, Southborough . T. C. Jones, Southborough Y. Konishi, Nara . D. Krewski, Ottawa· R Kroes, Bilthoven H. Luginbuhl, Bern . U. Mohr, Hannover . P. Olsen, Soborg J. A. Popp, Research Triangle Park· J. R Schenken, Omaha R A. Squire, Baltimore· J. Sugar, Budapest S. Takayama, Tokyo . G. C. Todd, Greenfield L. Tomatis, Lyon . B. F. Trump, Baltimore· J. M. Ward, Frederick

Officers - ILSI

Alex Malaspina, Atlanta - President Peter B. Dews, Boston - Vice President Ulrich Mohr, Hannover - Vice President Roger D. Middlekauff, Washington - Secretary/Treasurer

Respiratory System Edited by

T.e.Jones V.Mohr R.D.Hunt

With 279 Figures and 20 Tables

Springer-Verlag Berlin Heidelberg New York Tokyo 1985

Thomas Carlyle Jones, D. V. M., D. Sc. Professor of Comparative Pathology, Emeritus Harvard Medical School New England Regional Primate Research Center One Pine Hill Drive, Southborough, MA 01772, USA

Ulrich Mohr, M. D. Professor of Experimental Pathology Medizinische Hochschule Hannover Institut fur Experimentelle Pathologie Konstanty-Gutschow-Strasse 8 3000 Hannover 61, Federal Republic of Germany

Ronald Duncan Hunt, D. V. M. Professor of Comparative Pathology Harvard Medical School New England Regional Primate Research Center One Pine Hill Drive, Southborough, MA 01772, USA

ISBN-13: 978-3-642-96848-8 e-ISBN-13: 978-3-642-96846-4 DOl: 10.1007/978-3-642-96846-4

Library of Congress Cataloging in Publication Data. Main entry under title: Respiratory system. (Monographs on pathology oflaboratory animals) Bibliography: p. Includes index. 1. Laboratory animals-Diseases. 2. Respiratory organs-Diseases. 3. Rodents-Diseases. 4. Rodents as laboratory animals. 5. Pathology, Comparative. I. Jones, Thomas Carlyle. II. Mohr, U. (Ulrich) III. Hunt, Ronald Duncan. IV. Series. SF996.5.R47 1985 599.32'3 84-14048

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. Under § 54 of the German Copyright Law where copies are made for other than private use a fee is payable to 'Verwertungsgesellschaft Wort', Munich.

© Springer-Verlag Berlin Heidelberg 1985

The use of registered names, trademarks, etc. in the publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

Product Liability: The publisher can give no guarantee for information about drug dosage and application thereof contained in this book. In every individual case the respective user must check its accuracy by consulting other pharmaceutical literature.

2123/3140-543210

Foreword

The International Life Sciences Institute (ILSI) was created to promote cooperative efforts toward solving critical health and safety questions involving foods, drugs, cosmetics, chemicals, and other aspects of the environment. The Officers and Trustees believe that questions regarding health and safety are best resolved when government and industry rely on scientific investigations, analyses, and reviews by independent experts. Further, the scientific aspects of an issue should be examined and discussed on an international basis, separate from the political concerns of individual companies. ILSI is pleased to sponsor this set of monographs on the pathology of laboratory animals. This project will be useful in improving the scientific basis for the application of pathologic techniques to health and safety evaluation of substances in our environment. The world wide distribution of the authors, editors, and Editorial Board who are creating these monographs strengthens the expectation that international communication and cooperation will also be strengthened.

Alex Malaspina President International Life Sciences Institute

Preface

This book on the respiratory system is the second volume of a set prepared under the sponsorship of the International Life Sciences Institute (ILSI). One aim of this set on the Pathology of Laboratory Animals is to provide information which will be useful to pathologists, especially those involved in studies on the safety of foods, drugs, chemicals, and other substances in the environment. It is expected that this and future volumes will contribute to better communication on an international basis among people in government, industry, and academia who are involved in the protection of the public health. The arrangement of this volume is based, in part, upon the philosophy that the first step toward understanding a pathologic lesion is its precise and unambiguous identification. The microscopic and ultrastructural features of a lesion that are particularly useful to the pathologist for definitive diagnosis are therefore considered foremost. Diagnostic terms preferred by the author and editors are used as the subject heading for each pathologic lesion. Synonyms are listed although most are not preferred and some may have been used erroneously in prior publications. The problems arising in differential diagnosis of similar lesions are considered in detail. The biologic significance of each pathologic lesion is considered under such headings as etiology, natural history, pathogenesis, and frequency of occurrence under natural or experimental conditions. Comparison of information available on similar lesions in man and other species is valuable as a means to gain broader understanding of the processes involved. Knowledge of this nature is needed to form a scientific basis for safety evaluations and experimental pathology. References to pertinent literature are provided in close juxtaposition to the text in order to support conclusions in the text and lead toward additional information. Illustrations are an especially important means of non verbal communication, especially among pathologists, and therefore constitute important features of each volume. The subject under each heading is covered in concise terms and is expected to stand alone, but in some instances it is important to refer to other parts of the volume. A comprehensive index is provided to enhance the use of each volume as a reference. Some omissions are inevitable and we solicit comments from our colleagues to identify parts which need strengthening or correction. We have endeavored to include important lesions which a pathologist might encounter in studies involving the rat, mouse, or hamster. Newly recognized lesions or better understanding of old ones may make revised editions necessary in the future. The editors wish to express their deep gratitude to all of the individuals who have helped with this enterprise. We are indebted to each author and member of the Editorial Board whose names appear elsewhere in the volume. We are especially grateful to the Officers and Board of Trustees of the International Life Sciences Institute for their support and understanding. Several people have worked directly on important details in this venture. These include Nina Murray, Executive Secre-

VIII Preface

tary; Beverly Blake, Editorial Assistant; June Armstrong, Medical Illustrator; and Virginia Werwath, Administrative Assistant. Sharon K. Coleman, ILSI Coordinator for External Affairs, was helpful on many occasions. We are particularly grateful to Dr. Dietrich Gotze and his staff at Springer-Verlag for the quality of the published product.

November 1984 T.C.Jones V.Mohr R.D.Hunt

Table of Contents

The Upper Respiratory System (Nares, Larynx, Trachea) . 1

Histology, Ultrastructure, Embryology . . . . . . . . . . . . 3

Macrosopic, Microscopic, and Ultrastructural Anatomy of the Nasal Cavity, Rat J. A. POPP and N. A. MONTEIRO-RIVIERE ........... 3

Development of Syrian Golden Hamster Tracheal Epithelium During Prenatal and Immediate Postnatal Stages M.EMURA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Epithelial Alterations in Explant Cultures of Fetal Tracheae of Syrian Golden Hamsters M.EMURA. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 27

Neoplasms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Response to Carcinogens of Respiratory Epithelium, Syrian Golden Hamster (Mesocricetus Auratus) H.-B. RICHTER-REICHHELM, W. BONING, and J. ALTHOFF. 33

Polypoid Adenoma, Nasal Mucosa, Rat W.D.KERNS .................. .

Neoplasms, Mucosa, Ethmoid Turbinates, Rat S. F. STINSON and H. M. REZNIK-SCHOLLER .

Squamous Cell Carcinoma, Nasal Mucosa, Rat W.D.KERNS .................. .

Squamous Cell Carcinoma, Upper Respiratory Tract, Syrian Hamster

41

47

54

P. M. POUR. . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Adenocarcinoma, Anterior Nasal Epithelium, Rat S. F. STINSON and G. REZNIK ........... . 67

Hemangiosarcoma, Nasal Cavity, Mouse W. E. GIDDENS Jr. and R. A. RENNE .... 72

Clear Cell Carcinoma, Larynx, Syrian Hamster P.M. POUR .................... . 75

Lesions Due to Infections. . . . . . . . . . . . . . . . . . . . . .. 78

Murine Respiratory Mycoplasmosis, Upper Respiratory Tract, Rat T. R. SCHOEB and J. R. LINDSEY. . . . . . . . . . . . . . . . . 78

Sialodacryoadenitis Virus Infection, Upper Respiratory Tract, Rat D. G. BROWNSTEIN ....................... 84

X Table of Contents

The Lung (Bronchi, Bronchioles, Alveolar Ducts, Alveoli, Pleura) . 87

Histology and Ultrastructure . . . . .

Structure and Function of the Lung C. KUHN III ............ .

Neoplasms.

Bronchiolar/Alveolar Adenoma, Lung, Rat G. A. BOORMAN ............... .

Alveolar Type II Cell Adenoma, Lung, Mouse S. L. KAUFFMAN and T. SATO ........ .

Bronchiolar Adenoma, Lung, Mouse S. L. KAUFFMAN and T.SATO .....

Bronchiolar/Alveolar Carcinoma, Lung, Rat G. A. BOORMAN ................ .

Squamous Cell Carcinoma, Lung, Syrian Hamster P. M. POUR and H. M. REZNIK-SCHULLER.

Squamous Cell Carcinoma, Lung, Rat G. A. BOORMAN ............... .

Radiation-Induced Squamous Cell Carcinoma, Lung of Rodents

89

89

99

99

102

107

112

117

124

F. F. HAHN. . . . . . . . . . . . . . . . . . . . . . . . . . 127

Pleural Mesothelioma, Syrian Hamster A.CARDESAandJ.A.BOMBI ...

Metastatic Tumors, Lung, Mouse B. SASS and A. G. LIEBELT

Nonneoplastic Lesions . . .

Bleomycin-Induced Injury, Mouse: A Model for Pulmonary Fibrosis

133

138

160

D. H. BOWDEN .... . . . . . . . . . . . . . . . . . . 160

Endogenous Lipid Pneumonia in Female B6C3Fl Mice Y. EMI and Y. KONISHI ................. . 166

Pulmonary Lipidosis, Rat Y. EM I, R. HIGASHIGUCHI, and Y. KONISHI . 169

Alveolar Lipoproteinosis, Rat W.WELLER .................. . 171

Bronchiolar/Alveolar Hyperplasia, Lung, Rat G. A. BOORMAN ................ . 177

Fly Ash Pneumoconiosis, Hamster G. E. DAGLE and A. P. WEHNER 180

Asbestosis, Hamster G. E. DAGLE and A. P. WEHNER 183

Pulmonary Hair Embolism A. KAST ............ . 186

Table of Contents XI

Lesions Due to Infection . . . . . . . . . . . . .

Sendai Virus Infection, Lung, Mouse and Rat D. G. BROWNSTEIN ......... .

Rat Coronavirus Infection, Lung, Rat D. G. BROWNSTEIN ......... .

Pneumonia Virus of Mice Infection, Lung, Mouse and Rat

195

195

203

D. G. BROWNSTEIN ................. 206

Sialodacryoadenitits Virus Infection, Lung, Mouse D. G. BROWNSTEIN .............. .

Murine Respiratory Mycoplasmosis, Lung, Rat T. R. SCHOEB and J. R. LINDSEY ........ .

Pneumocystosis, Lung, Rat J. K. FRENKEL ......... .

Aspergillosis and Mucormycosis, Lung, Rat J. K. FRENKEL ............... .

Toxoplasmosis, Lung, Mouse and Hamster J. K. FRENKEL

Subject Index . .

210

213

218

224

227

231

List of Contributors

llirgen Althoff, M. D. Professor of Experimental Pathology, Hannover Medical School, 3000 Hannover 61, Federal Republic of Germany

Josep Antoni Bombi, M. D. Assistant Professor, Department of Pathology, University of Barcelona, Medical School, Barcelona, Spain

W. Boning, Dr. rer. nat. Hannover Medical School, Hannover, Federal Republic of Germany

Gary A. Boorman, D. V. M., Ph. D. Head, Tumor Pathology, Chemical Pathology Branch, NIEHS, Research Triangle Park, North Carolina, USA

Drummond H. Bowden, M. D. Professor and Head, Department of Pathology, University of Manitoba, Manitoba, Canada

David G. Brownstein, D. V. M. Associate Professor of Comparative Medicine, Yale University School of Medicine, New Haven, Connecticut, USA

A. Cardesa, M. D. Patologica Facultad de Medicina, Universidad de Barcelona, Barcelona, Spain

Gerald E. Dagle, D. V. M., Ph. D. Staff Pathologist, Battelle, Pacific Northwest Laboratory, Richland, Washington, USA

Yohko Emi, D.V.M. Department of Oncological Pathology, Cancer Center, Nara Medical College, Nara, Japan

Makito Emura, Priv. Doz. Dr. rer. nat. Head, Tissue Culture Unit, Institute of Experimental Pathology, Hannover Medical College, Hannover, Federal Republic of Germany

J. K. Frenkel, M. D., Ph. D. Professor of Pathology and Oncology, Department of Pathology and Oncology, University of Kansas Medical Center, Kansas City, Kansas, USA

W. Ellis Giddens, Jr., D. V. M., Ph. D. Associate Professor, Division of Animal Medicine, Department of Pathology, School of Medicine, University of Washington, Seattle, Washington, USA

Fletcher F. Hahn, D. V. M., Ph. D. Head, Pathology Group, Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Research Institute, Albuquerque, New Mexico, USA

Ryuichi Higashiguchi, M. D. Assistant, Department of Oncological Pathology, Cancer Center, Nara Medical College, Nara, Japan

XIV List of Contributors

Alexander Kast, Priv. Doz. Head, Department of Experimental Pathology, Nippon Boehringer Ingelheim Co. Ltd., Hyogo, Japan

Shirley L. Kauffman, M. S., M. D. Professor of Pathology, Department of Pathology, State University of New York, Downstate Medical Center, Brooklyn, New York, USA

William D. Kerns, D. V. M., M. S. Pathologist, Smith Kline & French Laboratories, Philadelphia, Pennsylvania, USA

Yoichi Konishi, M. D. Professor, Department of Oncological Pathology, Cancer Center, Nara Medical College, Nara, Japan

Charles Kuhn, III, M. D. Professor of Pathology, School of Medicine, Washington University, St. Louis, Missouri, USA

Annabel G. Liebelt, Ph. D. Biologist, Registry of Experimental Cancers, National Institutes of Health, Bethesda, Maryland, USA

J. Russell Lindsey, D. V. M., M. S. Professor, Department of Comparative Medicine, Univ. of Alabama, Director, Laboratory Animal Medicine, Veteran's Administration Medical Center, Birmingham, Alabama, USA

Nancy A. Monteiro-Riviere, M.S., Ph.D. Postdoctoral Fellow, Department of Pathology, C. I. I. T., Research Triangle Park, Visiting Assistant Professor, School of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, USA

James A. Popp, D. V. M., Ph. D. Head, Department of Experimental Pathology and Toxicology, Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina, USA

Parviz M. Pour, M. D. Professor, Eppley Institute for Research in Cancer, Department of Pathology and Laboratory Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USA

Roger A. Renne, D. V. M. Biology and Chemistry Department, Battelle, Pacific Northwest Laboratory, Richland, Washington, USA

Gerd Reznik, D. V. M., Priv. Doz. Pathology Services Project, National Center for Toxicological Research, Jefferson, Arkansas, USA

Hildegard M. Reznik-Schuller, D. V. M., Priv. Doz. Associate Professor of Experimental Oncology, Acting Chief, Laboratories of Experimental Therapeutics and Metabolism, NCI, Division of Cancer Treatment, Bethesda, Maryland, USA

H. B. Richter-Reichhelm, D. V. M. Priv. Doz. for Experimental Pathology, Max von Pettenkofer Institut, Bundesgesundheitsamt, Berlin, Federal Republic of Germany

Bernard Sass, D. V. M., M. S. Senior Investigator, Registry of Experimental Cancers, National Institutes of Health, Bethesda, Maryland, USA

List of Contributors XV

Tamiko Sato, M. D. Associate Professor of Anatomy, Department of Anatomy, New York Medical College, Valhalla, New York, USA

Trenton R. Schoeb, D. V. M., Ph. D. Assistant Professor of Comparative Medicine, Schools of Medicine and Dentistry, University of Alabama, Birmingham, Alabama, USA

Sherman F. Stinson, Ph. D. Frederick Cancer Research Center, Frederick, Maryland, USA

Alfrj;)d P. Wehner, D. M. D., D. D. S., Sc. D, cando med. Task Leader, Industrial Toxicology, Battelle, Pacific Northwest Laboratory, Richland, Washington, USA

W. Weller D. V. M. Silikose-Forschungsinstitut der Bergbau-Berufsgenossenschaft, Bochum, Federal Republic of Germany

The Upper Respiratory System (Nares, Larynx, Trachea)

HISTOLOGY, ULTRASTRUCTURE, EMBRYOLOGY

Macroscopic, Microscopic, and Ultrastructural Anatomy of the Nasal Cavity, Rat

James A. Popp and Nancy A. Monteiro-Riviere

For those interested in experimental studies of the nasal cavity, it is important first to understand the normal structure. This includes macroscopic, microscopic, and ultrastructural anatomic characteristics of the nasal cavity, surface epithelium, and submucosa. The medial surface of the three major turbinates is exposed when a midsagittal cut of a rat's nose is made and the septum is removed (Fig. 1). The nasoturbinate is located on the dorsal and anterior part of the nasal cavity, while the maxilloturbinate is located on the ventral and anterior part. The nasoturbinates and maxilloturbinates have relatively flat medial surfaces. The ethmoid turbinates consist of several lamellae : dorsal and ventral lamella of endoturbinate II, endoturbinate III, and a dorsal and ventral lamella of endoturbinate IV (Hebel and Stromberg 1976). Each endoturbinate has a flat medial surface and the more dorsal turbinate is larger than the ventral endoturbinates. Complete histological evaluation of normal turbinates requires examination of multiple levels to determine the distribution of epithelial types and submucosal glands and to determine variations within a single epithelial type based on location in the nasal cavity. Multiple sections are also re-

ABC 0

Fig.t. Schematic illustrations of the rat nasal cavity. Left: inverted rat skull with palatine structures as reference points for making cross sections ofthe nose. Right: sagittal

quired to identify and characterize lesions which may be localized in a small part of the nasal tissue. To achieve a uniform histological examination of the nasal passages, several groups have developed rather precise methods for making cross sections of the nose (Young 1981; Chang et al. 1983). One such uniform method is demonstrated in Fig.1. The blocks of tissue are hand cut with the previously decalcified skull inverted, and the location of the cross sections is determined by palatine structures. The first cut is made just anterior to the incisor teeth. The second cut is made halfway between the base of the incisor teeth and the incisive papilla. The third cut is made directly through the incisive papilla, while the fourth cut is made over the second palatal ridge, and the fifth cut is made through the second molar teeth. The resulting four blocks of tissue are embedded in either paraffin or glycol methacrylate with the anterior face down. Although this procedure provides a uniform sampling of the nasal structures, some alteration in the location of these sections may be necessary in specific experimental studies. The structures of the various nasal cavity surfaces at the locations defined above are indicated in Fig.2. At level A the nasoturbinate is attached to

section. Nasoturbinate (n), maxilloturbinate (m), and ethmoid turbinates (e). Lines indicate the location of sections taken for light microscopic examination

4 James A. Popp and Nancy A. Monteiro-Riviere

the dorsal lateral wall and extends a shorter distance into the nasal cavity in contrast to the section at level B. The maxilloturbinate is also less extensive and is attached to the ventral lateral wall of the nasal cavity. At level B, the nasoturbinate extends from the dorsal wall and projects ventrally to half the depth of the nasal cavity. Note that this turbinate turns laterally and dorsally, producing a hook in the cross section of turbinate. The maxilloturbinate is attached to the lateral wall and projects dorsally into the nasal cavity. The nasolacrimal duct lies ventral to it. In this section one can see the vomeronasal organ located in the ventral portion of the nasal septum. Although the function of this organ is still under investigation, it has a sensory function and is involved in pheromone-mediated behavior (Vaccarezza et al. 1981). At level C, the ethmoid turbinate appears to be free in the nasal cavity, since only the tip of the dorsal endoturbinate is included at this level. The nasoturbinates and max-

Fig. 2. A Transverse section through A, (Fig. 1) x 16; B transverse section through B, x 9; C transverse section through C, x 9; D transverse section through D, x 9. n, nasoturbinate; m, maxilloturbinate; e, ethmoid turbinate; s, nasal septum; w, lateral wall; v, vomeronasal organ; 0, location of septal olfactory organ; p, nasopharynx; d, nasolacrimal duct

illoturbinates are not present at this level. Since the palatine landmark for this section is through the incisive papilla, this structure is frequently observed on the palatine surface of the section. If the section is through the small nasal palatine ducts, stratified squamous epithelium is observed lining the ducts at the point where they connect the nasal and oral cavities. The fourth section (level D) is through the center of the ethmoid turbinate, which forms a complicated set of lamellae (scrolls) arising from the dorsal and lateral nasal walls. The nasopharynx is ventral to the! ethmoid turbinates. In specific virus-free rats, as defined by a standard rat murine viral antibody screening procedure (Microbiological Associates, Bethesda, Maryland), small lymphocyte accumulations are routinely found adjacent to this level of the nasopharynx, while leukocytes are not observed at other locations in the rat nasal mucosa. This small bit of lymphoid tissue adjacent to the nasopharynx is also seen consistently in mice.

Macroscopic, Microscopic, and Ultrastructural Anatomy of the Nasal Cavity, Rat 5

The nasal cavity is lined by three types of epithelium: squamous, respiratory, and olfactory. Squamous epithelium covers the nasal vestibule and the anterior tip of the nasoturbinate and maxilloturbinate and extends posteriorly as a narrow zone along the ventral nasal surface to the nasal palatine ducts. Respiratory epithelium covers all of the maxilloturbinate and most of the nasoturbinate except for its dorsal attachment, and also extends onto the anterior and ventral parts of the ethmoid turbinates. Olfactory epithelium covers the ethmoid turbinates, but also extends along the dorsal wall of the anterior nasal cavity to include the attachment of the nasoturbinate. The nasal septum is covered by respiratory epithelium except for some squamous epithelium in the area of the vestibule and olfactory epithelium on the dorsal attachment. A small oval area of olfactory epithelium exists on the ventral nasal septum just anterior to the septal window and is not contiguous with other olfactory epithelium. This focal area of olfactory epithelium is frequently referred to as the septal olfactory organ or the organ of Rodolfo-Masera, and may function as a detection mechanism during quiet respiration (Rodolfo-Masera 1943; Adams and McFarland 1971). The zones of demarcation between any two of the epithelial types are very abrupt, as is evident by either light or electron microscopy. Using morphometric procedures, the volume, total surface area, and surface area lined by each epithelial type have been quantitated for the nasal cavities of both rats and mice (Gross et al. 1982). In 16-week-old male Fischer-344rats, the nasal cavity has a volume of approximately 250 mm3

and a surface area of approximately 1350 mm2• In 16-week-old male B6C3Fl mice, the nasal volume is approximately 32 mm3 and the surface area is approximately 290 mm2• This large surface area is important in the warming, cleansing, and humidification of inspired air. Squamous epithelium covers 3% of the surface area in rats and 7% in mice, while the remainder of the surface is equally covered by respiratory (47% rats; 46% mice) and olfactory epithelium (50% rats, 47% mice). Detailed light microscopy of the surface epithelium of the nasal cavity has been completed. While no unique or surprising characteristics of the squamous epithelium have been described, interesting observations of the respiratory epithelium have been made. The nasal respiratory epithelium has been generally described as pseudostratified ciliated columnar epithelium. While this histological description is correct for the respiratory epi-

thelium found in some areas of the nasal cavity, other areas do not fit this general description. The respiratory epithelium in some areas, particularly the more anterior segments of the maxilloturbinates and nasoturbinates, consists of either cuboidal or nonciliated columnar cells, which may be found either alone or interspersed with a few ciliated cells (Fig. 3). Goblet cells are scattered unevenly throughout the respiratory epithelium and are most numerous in the nasal septum. In general, goblet cells are also relatively numerous in the ventral respiratory epithelium, particularly at the junction with squamous epithelium. Histologically, olfactory epithelium has a uniform pseudostratified columnar structure (Fig. 4). It is composed primarily of olfactory cells (bipolar neurons) and sustentacular cells, although a single row of basal cells is found adjacent to the basallamina. The intertwined cells make it impossible to distinguish individual cell borders. Nuclei are approximately six deep and covered with a nuclear-free zone of cytoplasm at the apical end. A thin eosinophilic zone composed of cilia and olfactory vesicles is present adjacent to the nasal cavity. A thin mucous layer is found on the surface of the olfactory and respiratory epithelium. The composition and function of this mucous blanket has been recently reviewed (Widdicomb and Wells 1982; Proctor 1982). The mucous layer consists of a superficial layer of mucus and an underlying watery periciliary fluid. The continuously moving layer is the first defense of the nasal cavity against inhaled gases and particles. Mucus is continually produced, flows on the nasal surfaces due to ciliary acitivity, and is ultimately swallowed after passing through the nasopharynx. The submucosal zone of the nasal passages is extremely vascular, although the vascularity is greatest in the nasoturbinates and maxilloturbinates. Between the numerous and relatively large vessels, 15-20 glands have been described in the submucosa of the septum, lateral wall, nasoturbinate, maxilloturbinate, and ethmoid turbinate (Bojsen-Moller 1964). The glands underlying respiratory epithelium are both serum and tp.ucus producing, with individual clusters of glandular tissue connected by ducts which pass anteriorly. Ducts of the serous glands reportedly empty into the vestibule of the nasal cavity (Bojsen-Moller 1964). The ducts open on inspiration and close on expiration, thereby releasing the glandular content of the serous glands only to incoming air to aid in the humidification of the air. Ducts of the mucous glands empty into the vomeronasal or-


Fig. 3 (Above). Light micrograph of respiratory epithelium. Cilliated (c) and non ciliated columnar (arrowhead) cells. Note the glands (g), duct (d), and blood vessel (b) in the submucosa. x 480

gan. In contrast to the different types of glands under the respiratory epithelium, the olfactory region has only a simple tubular mucus-producing gland (Bowman's gland), which opens directly on the surface (Bojsen-Meller 1964). Ultrastructural studies of toxin-induced lesions in the nasal mucosa have been published previously; however, the ultrastructural characteristics of the normal nasal structures had been incompletely described until recently (Monteiro-Riviere and Popp 1984). Transmission electron microscopic (TEM) studies of the respiratory epithelium dem-

Fig.4 (Below). Transition between olfactory (arrowhead) and respiratory epithelia. Bipolar neurons (n) and basal (b) and sustentacular (s) cells can be seen in the olfactory part, while ciliated (c) and goblet (g) cells can be seen in the respiratory portion. x 640

onstrated six distinct cell types: basal, cuboidal, nonciliated columnar, ciliated, brush, and goblet cells. The ciliated, basal, and goblet cells are similar to the comparable cell types described in other locations within the respiratory system. The cuboidal cell has sparse microvilli but no other distinctive ultrastructural characteristics (Fig. 5). The nonciliated columnar cell has an extensive accumulation of smooth endoplasmic reticulum in the apical cytoplasm (Fig. 6). The accumulation of this organelle suggests that nonciliated columnar cells may be the source of cytochrome P 450 and

Macroscopic, Microscopic, and Ultrastructural Anatomy of the Nasal Cavity, Rat 7

Fig.S (Above). A brush cell (B) and cuboidal cells (C) in respiratory epithelium lining the nasal cavity. Microvilli of brush cell protruding above (arrow) adjacent cuboidal cells. TEM, x 7400

Fig. 6 (Below). Two nonciliated columnar, cells (C) in respiratory epithelium. Microvilli (M) and extensive smooth endoplasmic reticulum (arrow) can bee seen in the apical region of the cell. TEM, x 14400 (Monteiro-Riviere and Popp 1984)


P450-associated enzymes that have been previously described in the nasal mucosa (Hadley and Dahl 1982). Unfortunately, information is not yet available on specific cell localization of P450 in the respiratory epithelium. The brush cell, with distinctive ultrastructural characteristics, has recently been described in the rat nasal respiratory epithelium (Monteiro-Riviere and Popp 1984). It is pear-shaped with a large basal part containing the nucleus, while the narrow apical surface extends into the nasal cavity (Fig. 5). Nonbranching microvilli cover the small surface. These microvilli are much longer and wider than microvilli of adjacent nonciliated cells, but are shorter than cilia. The apical cytoplasm has bundles of filaments and numerous clear vesicles. Paired cisternae are frequently seen in the supranuclear region. Although it has been hypothesized that brush cells in other locations may function as chemoreceptors, baroreceptors, or stretchreceptors (Meyrick and Reid 1968; Luciano et al. 1968, 1981), the function of this cell in the nasal cavity is unknown. In the rat nasal respiratory epithelium, intraepithelial nerve endings containing both clear and dense vesicles have been observed most frequent-

ly adjacent to the basal lamina (Fig. 7) (MonteiroRiviere and Popp 1984). They do not have a preferentiallocation adjacent to any specific cell type. The nerve endings and nerves in the respiratory epithelium and submucosa are branches of the trigeminal nerve and have a sensory function (Bojsen-M011er 1975). When examined by TEM, olfactory epithelium consists of three distinct cell types: sustentacular (supporting), olfactory (bipolar neuron), and basal cells (Frisch 1967). The bipolar neuron has an apical olfactory vesicle from which immotile cilia project in all directions. The sustentacular cell has long microvilli on the apical surface and pigment granules in the cytoplasm which account for the brown color of the olfactory epithelium noted upon gross observation. Scanning electron microscopy (SEM) of the normal nasal cavity clearly demonstrates that much of the surface is covered by a relatively smooth layer of mucus. When the mucous layer is removed, SEM allows one to study the surface characteristics of cells and determine the distribution of the various cell types on the basis of their surface structure. SEM dramatically demonstrates the uneven distribution of ciliated cells in the re-

Fig. 7. An intraepithelial axon (A) located just above basal lamina (BL) in respiratory epithelium. Clear vesicles (arrow), mitochondria, and neurotubules are present. TEM, x 33000 (Monteiro-Riviere and Popp 1984)

Macroscopic, Microscopic, and Ultrastructural Anatomy of the Nasal Cacity, Rat 9

Fig.8 (Above). Ciliated (C), nonciliated columnar (NC), and brush cell (arrowhead) in the respiratory epithelium. SEM, x 1800

Fig.9 (Below). Olfactory epithelium. Note the tangled web of cilia (arrowhead). SEM, x 4500


spiratory epithelium (J. A. Popp and J. T. Martin, unpublished work) (Fig. 8). In general, the anterior respiratory epithelium is nonciliated on the nasoturbinate, maxilloturbinate, and lateral wall. The surfaces become progressively more ciliated from anterior to posterior. However, the various surfaces are not equally ciliated at a single crosssectional level of the nasal cavity. For example, in the middle third of the nasoturbinate and maxilloturbinate, approximately 70% of the medial surface of the nasoturbinate is covered with ciliated cells while only 15% of the medial surface of the maxilloturbinate is covered with ciliated cells. In nonciliated areas, brush cells are easily identified due to their small surface area and long apical microvilli. They compose less than 1 % of the surface area, accounting for their infrequent observation in TEM studies. In contrast to the variable surface features in respiratory epithelium, SEM reveals the olfactory epithelium to be relatively uniform. The surface of the tissue is covered by a tangled web of cilia, although the tip of the olfactory vesicles may occasionally be observed in some locations (Fig. 9). A thorough understanding of the macroscopic, microscopic, and ultrastructural characteristics of the normal rodent nasal cavity has lagged behind experimental studies with nasal toxicants. A proper interpretation of lesions in the rat nasal cavity can be achieved only when one has a basic understanding of the normal nasal passages.

References

Adams DR, McFarland LZ (1971) Septal olfactory organ in Peromyscus. Comp Biochem Physiol (A) 40: 971-974

Bojsen-Moller F (1964) Topography of the nasal glands in rats and some other mammals. Anat Rec 150: 11-24

Bojsen-Moller F (1975) Demonstration of terminalis, olfactory, trigeminal and perivascular nerves in the rat nasal septum. J Comp Neuro1159: 245-256

Chang JC, Gross EA, Swenberg JA, Barrow CS (1983) Nasal cavity deposition, histopathology and cell proliferation after single or repeated formaldehyde exposures in B6C3F1 mice and F344 rats. Toxicol Appl Pharmacol 68: 161-176

Frisch 0 (1967) Ultrastructure of mouse olfactory mucosa. AmJ Anat 121: 87-120

Gross EA, Swenberg JA, Fields S, Popp JA (1982) Comparative morphometry of the nasal cavity in rats and mice. J Anat 135: 83-88

Hadley WM, Dahl AR (1982) Cytochrome P-450 dependent monooxygenase activity in rat nasal epithelial membranes. Toxicol Lett 10: 417-422

Hebel R, Stromberg MW (1976) Anatomy of the laboratory rat. Williams and Wilkins, Baltimore

Luciano L, Reale E, Ruska H (1968) Ueber eine 'chemorezeptive' Sinneszelle in der Trachea der Ratte. Z Zellforsch 85: 350-375

Luciano L, Castellucci M, Reale E (1981) The brush cells of the common bile duct of the rat. This section, freezefracture and scanning electron microscopy. Cell Tissue Res 218: 403-420

Meyrick B, Reid L (1968) The alveolar brush cell in rat lung - a third pneumonocyte. J Ultrastruct Res 23: 71-80

Monteiro-Riviere NA, Popp JA (1984) Ultrastructural characterization of the nasal respiratory epithelium in the rat. Am J Anat 169: 31-43

Popp JA, Martin JT (1984) Surface topography and distribution of cell types in the rat nasal respiratory epithelium: scanning electron microscopic observations. Am J Anat (in press)

Proctor OF (1982) The mucociliary system. In: Proctor OF, Andersen I (eds) The nose: upper airway physiology and atmospheric environment. Elsevier, New York, p245-278

Rodolfo-Masera DT (1943) Sui'esistenza di un particolare organo olfattivo nel sette nasale della cavia e di altri roditori. Arch Ital Anat Embriol48: 157-212

Vaccarezza OL, Sepich LN, Tramezzani JH (1981) The vomeronasal organ of the rat. J Anat 132: 167-185

Widdicomb JG, Wells UM (1982) Airway secretions. In: Proctor OF, Andersen I (eds) The nose: upper airway physiology and atmospheric environment. Elsevier, New York, p 215-224

Young JT (1981) Histopathologic examination of the rat nasal cavity. Fund Appl Toxicoll: 309-312

Development of Syrian Golden Hamster Tracheal Epithelium 11

Development of Syrian Golden Hamster Tracheal Epithelium During Prenatal and Immediate Postnatal Stages

Makito Emura

The Syrian golden hamster (Mesocricetus auratus) makes an excellent model for studies on chemical carcinogenesis of the respiratory tract, and in particular the trachea (Wynder and Hecht 1976). This organ is particularly sensitive to N-nitroso compounds, among others. It is also possible, using these compounds, to induce tumors transplacentally (Mohr 1973). For the study of the so-called early changes in animals exposed to strong chemical carcinogens, an understanding of the development of the trachea is necessary (Mohr et al. 1979). The anlage of the trachea in the fetus cannot be easily distinguished until after the 9th day of pregnancy, but only a few days later signs of rapid growth and differentiation are clearly recognizable.

Predifferentiation Stage

Light Microscopy. The tracheobronchial rudiments of the Syrian hamster become independent of the early esophageal ducts (Fig. 10) between the 9th and 10th gestational days. The next stage, extending from the 10th to 11th gestational days, can be regarded as the predifferentiation stage, since no marked sign of differentiation is detected

Fig. to (Left). Longitudinal sections of tracheal epithelium, Syrian hamster on the 10th gestational day. Trachea (T) and esophagus (OE) with ventral (top) and dorsal epithelium (bottom). H, heart. Hand E, x 43

in the epithelium either by light or electron microscopy. At this stage the tracheal epithelium is mainly composed of one layer of tall and narrow columnar cells (Fig. 11); the nuclei are elongated, ovoid, or round and basally situated. At the luminal surfaces, the epithelium also contains a few ellipsoid or polygonal cells. Several cells possess cytoplasmic vacuoles. In a relatively few epithelial cells, the luminal portion of the cytoplasm is PAS positive. When pretreated with diastase, very few cells subsequently react to PAS and none stain with alcian blue.

Electron Microscopy. At this stage no signs of differentiation can be detected in the cells and no particular cell types are discernible (Fig. 12). The irregular luminal surface usually possesses sparse cytoplasmic projections or microvilli of various lengths (Figs. 13 and 14). The nuclei are round to ovoid and their contours are mostly smooth and usually contain two to four nucleoli. The nuclear chromatin fibrils are uniformly dispersed in the nucleoplasm of epithelial cells and none of the chromatin condensations of the type seen in maturing cell types are recognizable until the 12th day. In the stromal fibroblasts, however, such chromatin condensations are already beginning

Fig.11 (Right). Trachea at higher magnification. Hand E, x 106

12 Makito Emura

Fig.12 (Upper left). Epithelial cells on the 11th gestational day. Note sparse endoplasmic reticulum (ER), abundance of free polyribosomes and glycogen granules (G), uniformly diffuse nuclear chromatin fibrils, smooth contours of nuclei, distinct nucleolonemas of the nucleoli (NL), and intercellular spaces. In comparison with the 10th gestational day, the only difference is the absence of cytoplasmic vacuoles and vesicles. Centrioles (C) and small cytoplasmic projections can be seen. TEM, uranyl acetate and lead citrate, x 5070

Fig.13 (Lower left). Epithelial cell on the 11th gestational day. Note the small amount of ER and glycogen (G). TEM, x 11640

Fig. 14 (Upper right). a Vesicles in the epithelial cells on the 10th gestational day. Note relatively large polyribosomes in the cytoplasm and vesicles (arrow), which begin to resemble ER. TEM, x 19890. b Accumulation of glycogen (G) can be observed around the protrusions and vesicle membrane. Arrow indicates ribosomes attached to membrane. TEM, uranylacetate and lead citrate, x 11230

Fig. 15 (Lower right). A solitary immature cilium projecting from the luminal surface. TEM, uranyl acetate and lead citrate, x 11640


to occur. Glycogen granules are either scattered throughout the cytoplasm or accumulate in a small part of the cytoplasm (Fig. 13). Some cells possess one or two cilia (Fig. 15). In the luminal part of the cytoplasm a few centrioles can occasionally be observed (Figs.12 and 15) and peculiar tightly bound intercellular junctions are formed directly against the lumen (Figs.12 and 13). The mitochondria are round to elongated, frequently club-shaped, and their matrices contain dense or sparse fine fibrillar or granular materials. Free polyribosomes prevail. Distinct but not well-developed Golgi apparatus assume mainly lamellar structures with some vesicles, mostly located near the nuclei (Fig. 13). The basement membrane is distinct and further deposition of fibrillar material progresses. On the 10th gestational day, the smooth and rough endoplasmic reticulum (ER) of the epithelial cells seem poorly developed and in most cases flattened sac or saccule forms are found. The outer nuclear membrane in these cells very frequently has widely distributed extranuclear protrusions (Emura 1978) and often contains membranous structures. On the 11th gestational day, the smooth and rough ER increases only slightly (Fig. 13) and the frequency and size of the protrusions extending from the outer nuclear membrane decrease remarkably. The rough- and smooth-membranebound vacuoles and vesicles also notably de-

Fig. 16. Longitudinal section of tracheal epithelium. Cranial ventral section. A cartilaginous mesenchyme condensate (M), cells with hemispherical luminal apices and basophilic cytoplasm (black arrows), and basally situated cells (white arrows) are shown. Hand E, x 170

Fig.17. Longitudinal section of tracheal epithelium. Caudal, dorsal section. Note cell with flat luminal surface and eosinophilic cytoplasm (thick arrow). Hand E, x 170

crease in number. However, around the periphery of the nuclei, peculiar intranuclear membranous inclusions appear at intervals and their frequency increases as time progresses. The possibility that these vacuoles and vesicles contribute to the formation of ER cannot be excluded, since in vertebrate and invertebrate oocytes (Wischnitzer 1974) and in embryonic epithelium of chick choroidal plexus (Birge and Doolin 1974) the rough ER has been demonstrated to originate in vesicles derived from the outer nuclear membrane.

Early Morphological Indication of Differentiation

Light Microscopy. On the 12th gestational day, the epithelial cells are somewhat flatter than those seen on previous days. No distinct cell types are detected by light microscopy. On the 13th gestational day, approximately 20 horseshoe-shaped cartilaginous condensates consisting of mesenchymal cells are formed in the trachea (Fig.16). In the epithelium, three cell types can be distinguished. These are tentatively designated as type I, type II, and type III cells in this report. The type I cells feature a hemispherical luminal apex protruding into the lumen, and are characterized by a somewhat basophilic or less eosinophilic cytoplasm (Fig.16). Flat luminal surfaces and somewhat eosinophilic or less basophilic

14 Makito Emura

cytoplasm characterize the type II cells (Fig. 17). Cells of this type are most prominent in the dorsal epithelium and are not positive to PAS. The type III cells are basally situated and have oblong, triangular, or polygonal shapes (Fig. 16). The presence of type I and type III cells causes the epithelium to assume a double-layered appearance in

Fig.1S (Above). Luminal cytoplasm of cells with hemispherical surfaces (type I) and cells with flat luminal surfaces (type II). Note the abundance of rough ER. Dictyosome (DCT). TEM, uranyl acetate and lead citrate, x 11640

Fig.19 (Lower left). Luminal part of a type II cell on the 13th gestational day. Note fibrillar material interspersed with dark granules, 21 x 21 to 87 x 114 nm in size. TEM, x 22770

parts, i. e., a luminal and basal layer. Several epithelial cells react positively to PAS, but after pretreatment with diastase practically no cell has a positive reaction to PAS.

Electron Microscopy. On the 12th gestational day, the luminal apices in most epithelial cells pro-

Fig. 20 (Lower right). Luminal part of a cell resembling type I, on the 13th gestational day. Fibrillar material interspersed with dark granules can be seen (26 x 53 to 42 x 65 nm). Note the proximity of the fibrillar material to existing centriole. TEM, uranyl acetate and lead citrate, x 22770


trude slightly into the lumen, although their luminal surfaces are still irregular. The scattered glycogen granules have diminished in many of the cells; the ER is not well developed, possesses rough surfaces, and assumes flattened sac-like forms. The outer nuclear membrane again starts to form circumscribed extensions, which are small but similar to those observed on the 10th gestational day. Membranous and vesicular intranuclear inclusions at the nuclear periphery occur more frequently than on the 11 th gestational day. The smooth- and rough-membrane-bound vacuoles and vesicles already seen on the 10th gestational day occur again, although only occasionally. The nuclei are still round to ovoid in shape with smooth contours. Distinct condensations of nuclear chromatin fibrils occur in 20%-30% of epithelial cells along the nuclear envelope, as well as in the inner area of the nuclei, although this is much less extensive than in maturing fetal mucous cells. On the 13th gestational day, three cell types can be distinguished in the epithelium. The first type

Fig.21 (Upper left). Longitudinal sections of the ventral epithelium (pars cartilaginea). No ciliated cel\s are seen. Cytoplasmic vesicles are prominent. Hand E, x 170

Fig. 22 (Lower left). Longitudinal sections of dorsal epithelium (pars membranacea). A few obviously ciliated cel\s

(I) is composed of cells with smooth hemispherical luminal surfaces protruding into the lumen (Fig. 18). The second type (II) consists of cells with flat luminal surfaces, on which several short microvilli or cytoplasmic projections can be observed (Figs. 18 and 19). The ventral and lateral epithelial cells are largely composed of type I cells. In the dorsal epithelium, type II cells seldom occur; type I cells still predominate. Cells of both types often contain one or two regional accumulations of a considerable amount of vesicular and tubular smooth ER in the luminal apices (Figs. 18 and 20). With these accumulations of smooth ER, frequently found in both cell types, dictyosomes develop which are composed of three to seven cisternae (Figs. 18 and 20). In the luminal cytoplasm, the rough ER and free polyribosomes are frequently more abundant in the type I than in the type II cells. However, the basal cytoplasm of both cell types apparently contains the same amount of rough ER. In a small number of type I cells, fairly numerous ER vesicles surrounded by a partly rough membrane can be observed. Generally, in both types of cells, rough ER increases only fractionally in compari-

(C), type II cel\s (II) , and type III cel\s (III) classified on the 13th gestational day. Hand E, x 170

Fig.23 (Right). Longitudinal section of cranial part of the ventral tracheal epithelium on the 16th gestational day. Note ciliated cel\ (arrow). Hand E, x 170

16 Makito Emura

son with previous days of development. Connection of smooth ER with Golgi (or dictyosome) cisternae is occasionally encountered. Located near the smooth ER accumulations and dictyosomes sometimes found in type II cells are small areas of fine fibrillar material, often interspersed with a few dark granules, ranging from 21 x21 to 87-114nm (average 49x65nm) in size. These are identified as structures similar to "proliferative elements" (Dirksen and Crocker 1966) (Figs. 19 and 20), and they also occur in the luminal cytoplasm of type I cells, although much less frequently. The inner sections of this fibrillar material are usually devoid of ribosomes. The third type (III) of cells are basally situated in the epithelium and resemble type I cells, except that they have no free luminal surfaces. In cells of all three types, as well as the stromal

fibroblasts, a few immature-looking cilia and centrioles occasionally occur. On the 13th gestational day, the circumscribed extensions of outer nuclear membrane in the trachea, also encountered in the three epithelial cell types and in stromal fibroblasts, are more frequent and more conspicuous than on the previous day. Their frequency and size are similar to those of the 10th gestational day. The vesicular and tubular intranuclear inclusions are also more frequent and more conspicuous than on the 11th and 12th days. However, the rough- and smooth-membrane-bound vacuoles and vesicles, which persistently occur, are not so frequent as in the differentiating type II cells on the 14th gestational day. These vacuoles and vesicles appear to fuse occasionally with the preexisting rough ER.

ig.24 (Above). Type II (ciliated) cell on the 14th gestational day. ote ab ence of rough ER. Uranyl acetate and lead citrate x 16640

Fig.25 (Below). Higher magnification of Fig. 24. Procentrioles (PC): "conden ation form" (CF). Uranyl acetate and lead citrate. x 31590


Golgi cisternae are sometimes connected to the nuclear envelope. Condensation of nuclear chromatin occurs more extensively on the 13th gestational day in 80%-95% of cells of all types, including the stromal fibroblast.

Differentiation of Ciliated Cells

Electron microscopy reveals that the primary stage of ciliogenesis takes place on the 13th gestational day in some of the type II cells. However, the first ciliated cells can be detected on the 14th gestational day in the dorsal epithelium (pars membranacea) by both light and electron microscopy.

Light Microscopy. The epithelium on the 14th gestational and following days of development also has a double-layered appearance. The ciliated cells possess granular eosinophilic cytoplasm and flat luminal surfaces, which are features similar to those of the type II cells of the 13th gestational day (Figs.21 and 22). At the caudal part of the dorsal epithelium these cells are usually cylindricalor cuboidal, sometimes oblong, and extend from the basement membrane to the lumen. In the cranial part of the epithelium, they often assume either conchoidal or bell shapes. Ciliated cells are sparse in the ventral and lateral epithelium (pars cartilaginea). In most of the trachea, the cilia are often longer in the cranial part than in the caudal part of the epithelium. The type II cells observed on the 13th gestational day (Fig.17) are still encountered frequently on the 14th gestational day, particularly in the middle to caudal part of the dorsal epithelium. On the 15th and 16th gestational days, ciliated cells are only found sporadically in the ventral and lateral epithelium (Fig.23). On the last gestational day, the cells are well developed in the dorsal epithelium but occur less frequently in the ventral and lateral epithelium.

Electron Microscopy. On the 14th gestational day, the three cell types distinguished on the previous day develop more distinct features. On this day the type II cells are at various stages of organellic differentiation. They possess flat luminal surfaces and occur more frequently in the dorsal epithelium than on the previous day. A small amount of smooth ER and a few ribosomes exist; these can occasionally be seen in cells of the same type on the previous day. Some cells are seen in various stages of ciliogenesis (Figs. 24-31).

In cells at an early stage of ciliogenesis, the fibrillar material alone or with the dark granules, 39 x 42 to 73 x 96 nm (average 53 x 66 nm), occurs more frequently than on the 13th day. Among these granules much darker bodies, 63-148 nm in diameter, or larger hollow bodies, 148-208 nm and 63 -1 04 nm in outer and inner diameters respectively, are often identified as "condensation forms" (Dirksen and Crocker 1966) (Figs. 24-29). Tubular and vesicular structures are often located near areas of dark granules (Figs.27-30). In cells at a different stage of ciliogenesis, several procentrioles, measuring between 116 x 127 and 158 x 180 nm, are associated with the condensation forms. In more advanced cells, almost complete centrioles measuring 180-380 nm and smaller, denser condensation forms 48-95 nm in diameter occur (Fig. 29). These centrioles, which eventually become ciliary basal bodies, are formed by a process which Anderson and Brenner (1971) termed "acentriolar basal body formation," in which the centrioles develop together with structures which bear no resemblance to centrioles. The process of ciliogenesis described here seems to correspond to this theory. In developing fetal rats (Stockinger and Cireli 1965; Dirksen and Crocker 1966) and mice (Frisch and Farbman 1968) such fibrogranular material has been reported. Stockinger and Cireli (1965) suggested that this fibrogranular material was formed de novo without any influence of preexisting mature centrioles and that the granular materials which were considered to be precentrioles developed through various intermediate stages into mature centrioles. Dirksen and Crocker (1966) found a direct link between mature centrioles and this fibrogranular material and termed them "proliferative elements." In the fetal rat, Sorokin (1968) suggests a similar process in which "deuterosomes" seem to correspond to the condensation forms. Another possibility is that the preexisting centrioles may be decondensed into the fibrillar material which would function later as templates for new microtubule proteins (Dirksen and Crocker 1966; Staprans and Dirksen 1974). In cells at a somewhat later stage of ciliogertesis, complete centrioles possessing nine triplets of microtubules accumulate in the luminal apices (Fig. 30). The cells undergoing ciliogenesis usually possess well-developed microvilli. Glycogen granules are often absent from the type II and ciliated cells. In the developing Syrian hamster trachea the fibrillar material with dense granules can first be detected on the 13th gestational day, and

18 Makito Emura

Fig.26 (Upper/eft). Type II (ciliated) cell on the 14th gestational day. Uranyl acetate and lead citrate, x 22770

Fig.27 (Upper right). Higher magnification of Fig. 26. "Growing condensation forms" (GCF) with hollow center; a solitary condensation form (CF) and a ciliary bud-like tubule (arrow) procentriole (PC). Uranyl acetate and lead citrate, x 47320

Fig. 28 (Below). Type II (ciliated) cell on the 14th gestation· al day. Various structures related to ciliogenesis: fibrogran· ular material (FM); centriole (C); condensation forms (CF); procentrioles (PC). Uranyl acetate and lead citrate, x 47320


Fig.29. Type II cell. Centrioles near maturation and condensation forms (CF). Uranyl acetate and lead citrate, x 31590

Fig.31. Cilia growing from centrioles in type II cells on the 14th gestational day. Uranyl acetate and lead citrate x 16640

only on the following day do ciliated cells occur. Therefore, it can be supposed that a period of 1 day is sufficient for completion of the successive stages preceding ciliogenesis. From the 14th gestational day onward, ciliogenesis occurs in an increasing number of the type II cells. On the 1st postnatal day, typical mature cil-

Fig.30. Type II cell. Almost mature centrioles. Note the microtubule triplets in one cross section (arrow) and the centriole-associated vesicles (CV). Uranyl acetate and lead citrate, x 31590

iated cells are frequently observed (Fig. 32). However, ciliogenesis on the 14th and subsequent gestational days is not always restricted to type II cells, but also occurs in cells apparently of type I at various stages of differentiation of secretory systems, although much less frequently.

20 Makito Emura

Fig.33 (Left). Dorsal epithelium on the 15th gestational day. Note cell with pale cytoplasm (arrow). Hand E, x 170

Differentiation of Mucous Cells

Light Microscopy. On the 14th gestational day type I and type II cells are prominent in the epithelium (Figs.21 and 22). Their cytoplasm often appears vacuolated. The epithelium of the 15th and 16th gestational days resembles that of the 14th gestational day. However, the luminal apices of the type I cells are no longer hemispherical but slightly protruded and round. Although occurring at a low frequency, cells with pale cytoplasm between the luminal surface and nucleus appear in the cranial part of the epithelium (Fig. 33). On the last gestational day, the epithelial cells with pale cytoplasm (Fig.33) are more numerous and the first mature mucous cells are observed (Fig. 34).

Fig.32. Mature ciliated cell on the 1st postnatal day. Uranyl acetate and lead citrate, x 11 640

Fig.34 (Right) . Ventral epithelium, 15th gestational day. A few mucous cells with pale cytoplasm. Hand E, x 170

The number of cells that react positively to PAS sharply increases around the 14th gestational day (Emura and Mohr 1975), and continues to increase during the next 4 weeks of the postnatal period. Notably, even the type III cells react positively to PAS, particularly around the nuclei. After diastase pretreatment, however, the positive PAS reaction disappears from around the nuclei of many cells, especially from those of the basal layer (type III). On the 14th gestational day, very few cells react to PAS following diastase, but around the 16th gestational or 1st neonatal day the frequency of such cells increases (Emura and Mohr 1975). In these cells only the cytoplasmic portion between the luminal surface and nucleus reacts positively to


PAS. With progressive development, these areas acquire more and larger granules, expand, and finally occupy the entire cytoplasm above the nucleus. Many differentiating type I cells contain PAS-positive material in the cytoplasm above the nucleus. Such material greatly diminishes in cells toward the caudal part of the epithelium (Fig. 35). Few cells stain with alcian blue until the 14th gestational day. From this day onward they remain at an average level of 13% of epithelial cells, which is approximately half the average frequency of PAS-positive cells that resist diastase treatment. Throughout these developmental stages, the ma-

Fig. 35 (Above). Ventral epithelium on the 1st postnatal day. Diastase and PAS, x 106

Fig.36 (Middle). Epithelium on the 1st postnatal day, dorsal surface. Cells positive to alcian blue are dark. Alcian blue, x 105

Fig.37 (Lower left). Dorsal epithelial cells on the 1st postnatal day. Cytoplasm near lumen stains with alcian blue. Alcian blue and nuclear red, x 425

jority of cells that stain with alcian blue also react to PAS after diastase, but the reverse is not true (Emura and Mohr 1975). On the 14th and 15th gestational days the cytoplasm of some cells stains with alcian blue around the nucleus and just beneath the luminal surface. These cells occur more frequently in the cranial to middle parts of the dorsal epithelium on the 16th gestational to the 1st postnatal day (Figs.35 and 36). At these stages, cells stain with alcian blue, mainly in the cytoplasmic portion between the luminal surface and the nuclei (Figs. 36 and 37). No marked difference can be detected in the frequency of cells reacting to PAS and alcian blue

Fig.38 (Lower right). Tracheal epithelium on the 1st postnatal day. On the left is a ciliated cell; in the center are two cells which, following diastase, have PAS-positive material in their cytoplasm. The cell in the center is ciliated; two on the right have spiny processes on the luminal side. PAS and hematoxylin, x 425

22 Makito Emura

Fig.39 (Upper left). Hamster trachea, 13th gestational day. Type I epithelial cell with partly rough vesicles resembling ER. Uranyl acetate and lead citrate, x 11640

Fig.40 (Lower). Differentiating type I cells on the 14th gestational day. Cells at two different stages of differentiation. In the cell MCll, the winding cisternae of rough ER which contain dense material are prominent. There already exists a small amount of vesicular ER. The cells MCI seem to be at the same stage as those in Fig.41. The extensions and the local cisternal dilation of the nuclear envelope are no long-

er detectable in the cell Men, while in Mel tlley are both conspicuous (arrows). Note the partly rough membrane portion of the vesicles in Mel. The cell Men apears to be at a later stage of differentiation than the cell Mel. Uranyl acetate and lead citrate, x 11640

Fig.41 (Upper right). Type I (mucous) cells on the 14th gestational day. Vesicles are enclosed by a partially granular membrane. Note chromatin condensation, focal cisternal dilation of the nuclear envelope, and glycogen granules (G). Uranyl acetate and lead citrate, x 11640


between the prenatal and the neonatal epithelium on the 16th day. Some ciliated cells on the 16th gestational and the 1st postnatal days clearly react to PAS even after diastase pretreatment (Fig. 38). A few also stain with alcian blue.

Electron Microscopy. In type I cells rough ER noticeably increases on the 14th -16th gestational days. It can therefore be presumed that type I cells eventually become mucous cells. However, the ultrastructure of the rough ER has several different aspects, depending on the stage of differentiation of the cells. In some of the primitive type I cells at the primary stage (Figs.39 and 41), the vacuoles and vesicles possessing both smooth and rough areas begin to resemble ER. These structures are found in blastic nerve cells (Pannese 1968), in developing pancreatic cells (Wessells and Evans 1968), and in embryonic choroidal epithelial cells (Birge and Doolin 1974). The nuclear envelope of these primitive type I cells usually undergoes local cisternal dilation and/ or circumscribed extensions of the outer membrane. This is most frequent and prominent on the 14th gestational day. In differentiating type I cells at advanced stages, almost all of the ER is rough and its flattened winding cisternae are extended and contain electron-dense, fibrillar, and amorphous material (Fig.40). In these type I cells at more advanced stages, rough and smooth vesicular ER occur in addition to the flattened, winding ER. The cisternae of this vesicular ER usually contain fibrillar or amorphous material. Thus, in time, an increasing number of type I cells with cisternae of rough ER and/or vesicles limited by smooth and rough ER membranes, all containing electron-dense, fibrillar, and amorphous material, fill the entire cytoplasm (Fig. 40). At the same time, dictyosomes of Golgi apparatus develop extensively (Figs.40 and 42). Finally, on the 1st postnatal day, maturing mucous (type I) cells appear (Figs.43 and 44). In these cells the nuclear chromatin is more condensed than that of the mature ciliated cell (Fig. 32). The cisternae of the nuclear envelope are usually locally dilated in many areas (Fig. 44), and the outer nuclear membrane frequently comes into contact with the cytoplasmic vesicles. Some vesicles contain less electron-dense material than others. Both types of of vesicles frequently fuse together in the luminal cytoplasm (Fig. 44). This kind of mucous cell is presumably of the neonatal type, since it occurs only during the neonatal period and is not found in the adult epithelium. On the 1st postnatal day, cells with well-developed

rough ER and Golgi apparatus at various stages of differentiation are still abundant (Fig. 45). From the 14th gestational up until the 1st postnatal day, many of the epithelial cells, including basal cells, contain glycogen granules (Figs.40 and 42). Frequent association of glycogen granules with protrusions of the outer nuclear membrane would suggest that some of these membranes play a part in glycogen metabolism similar to the function of smooth ER of liver parenchymal cells (Coimbra and Leblond 1966). After the 15th gestational day, another type of cell which appears to be secretory in nature occurs (Figs.43 and 46), although its origin is unclear. The cells lack the typical rough ER that synthesizes secretory protein. Instead, they contain vacuoles and vesicles surrounded by smooth and rough membranes. As observed by light microscopy, after the 14th gestational day cells frequently undergo ciliogenesis (type II) and produce mucus (type I). In prospective mucous cells, smooth- and roughmembrane-bound vesicles exist, together with proliferating centrioles (Fig.48). Other such cells possess several cilia, Golgi apparatus, and rough ER, which develop to a considerable extent (Fig.47). In cells which basically resemble the secretory cells shown in Fig.46 and which lack granular ER, large smooth- and rough-surfaced vesicles are found together with cilia or proliferated centrioles (Fig.49). However, it is usual in all these types of cells to find that the centrioles, cilia, rough ER, and cytoplasmic vesicles develop poorly compared with the same organelles in differentiating type I or type II cells. In the mature mucous cells no centrioles or cilia can be detected. Cells containing both mucous granules and cilia have been reported in the respiratory epithelium of neonatal rats (Stockinger and Cireli 1965), adult Syrian hamsters in regeneration (McDowell et al. 1979), and adult humans (McDowell et al. 1978).

Changes in Mitotic Activity. In the longitudinally cut hamster tracheal epithelium, the mitotic indices are counted in percentages (see Emura and Mohr 1975). The highest percentage of 4.50/0 is seen on the 11 th gestational day during the predifferentiation stage. The index sharply declines on the 12th gestational day (2.5%), and then continues to decline until the 14th gestational day (1.8%), when the first ciliated cells can be observed. Following this, a more gradual decrease is noticed on the 1st postnatal day (0.3%), when mature mucous cells are first observed.

24 Makito Emura


<1 Fig.42 (Above). Luminal cytoplasm of a differentiating type I cell on the 16th gestational day. Vesiculated ER cisternae limited by a rough membrane are especially prominent in the cytoplasm near the lumen. Note the well-developed Golgi apparatus and the vesiculated, partly rough ER cisternae. Uranyl acetate and lead citrate, x 22770

Fig.43 (Below). Epithelium, 1st postnatal day, dorsal aspect. Cells in various stages of differentiation. Uranyl acetate and lead citrate, x 5070

Fig. 44 (Above). Type I cell on 1st postnatal day. Nearly mature mucous cell with vesicles of low and high density. In vesicles of high density the limiting membrane is difficult to distinguish. Note the fusion of the two different types of vesicles (arrows). Golgi apparatus (GA). Uranyl acetate and lead citrate, x 16640

Fig.45 (Below). Type I cells in dorsal cranial epithelium on 1st postnatal day. The cell MCIV seems to be at the same stage as that of the cell in Fig.42. The cell MCVI appears to be developing into the cell shown in Fig. 44. Uranyl acetate and lead citrate, x 11640

26 Makito Emura

Fig.46 (Upper left). Secretory cell on the 1st postnatal day. Uranyl acetate and lead citrate, x 11640

Fig.47 (Lower left). A differentiating type I cell (left) and a cell between a mucous and ciliated cell (right), on the 1st postnatal day. Note that the state of the chromatin condensation of the cell on the right appears to be at a stage between the ciliated and the type I cell. Uranyl acetate and lead citrate, x 11 640

Uneven Differentiation Pattern of Ciliated and Mucous Cells. The first ciliated cells occur in the dorsal epithelium (pars membranacea) of the trachea on the 14th gestational day. Ciliated cells are virtually absent in the ventral and lateral epithelium

Fig.48 (Upper right). A cell in the stage between ciliogenesis and mucogenesis on the 14th gestational day. Centrioles and partly rough vesicles are prominent. Uranyl acetate and lead citrate, x 7740

Fig.49 (Lower right). A cell in a stage of differentiation between the ciliated cell and the presumed secretory cell in Fig.46. The chromatin condensation is similar to that of the ciliated cell (Fig.32). Uranyl acetate and lead citrate, x 11640

(pars cartilaginea), and at this stage occur more frequently and are longer in the cranial than in the caudal part of the dorsal epithelium. On the 15th gestational day, the cells also appear in the ventral and lateral epithelium and thereafter increase

Epithelial Alterations in Explant Cultures of Fetal Tracheae of Syrian Golden Hamsters 27

gradually in number. Even on the 1st postnatal day they are seen more frequently in the dorsal than in the ventral and lateral epithelium. The cells which are resistant to diastase treatment and react positively to PAS and those cells that stain with alcian blue are unevenly distributed along the ventral and dorsal epithelium during the developmental stages (Figs.35 and 36). Both of these cell types occur primarily in the cranial part of the epithelium. As development progresses, they gradually appear in the caudal part of the epithelium. From the 14th to the 16th gestational days, they occur more frequently in the dorsal than in the ventral epithelium. On the 1st postnatal day, this difference is no longer evident.

References

Anderson RGW, Brenner RM (1971) The formation of basal bodies (centrioles) in the rhesus monkey oviduct. J Cell BioI 50: 10-34

Birge WJ, Doolin PF (1974) The ultrastructural differentiation of the endoplasmic reticulum in choroidal epithelial cells of the chick embryo. Tissue Cell 6: 335-360

Coimbra A, Leblond CP (1966) Sites of glycogen synthesis in rat liver cells as shown by electron microscope radioautography after administration of glucose-H3. J Cell BioI 30: 151-175

Dirksen ER, Crocker IT (1966) Centriole replication in differentiating ciliated cells of mammalian respiratory epithelium. An electron microscopic study. J Microscop 5: 629-644

Emura M (1978) Morphological studies on the development of tracheal epithelium in the Syrian golden hamster. IV. Electron microscopy: blebbing of nuclear membrane. Z Versuchstierkd 20: 163-170

Emura M, Mohr U (1975) Morphological studies on the development of tracheal epithelium in the Syrian golden hamster. I. Light microscopy. Z Versuchstierkd 17: 14-26

Frisch D, Farbman AI (1968) Development of order during ciliogenesis. Anat Rec 162: 221-232

McDowell EM, Barrett LA, Glavin F, Harris CC, Trump BF (1978) The respiratory epithelium. I. Human bronchus. JNCI 61: 539-549

McDowell EM, Becci PJ, Schurch W, Trump BF (1979) The respiratory epithelium. VII. Epidermoid metaplasia of hamster tracheal epithelium during regeneration following mechanical injury. JNCI 62: 995-1008

Mohr U (1973) Effects of diethylnitrosamine on fetal and suckling Syrian golden hamsters. IARC Sci Publ 4: 65-70

Mohr U, Reznik-Schuller H, Emura M (1979) Tissue differentiation as a prerequisite for transplacental carcinogenesis in the hamster respiratory system, with specific respect to the trachea. Nat! Cancer Inst Monogr 51: 117-122

Pannese E (1968) Developmental changes of the endoplasmic reticulum and ribosomes in nerve cells of the spinal ganglia of the domestic fowl. J Comp Neurol 132: 331-364

Sorokin SP (1968) Reconstructions of centriole formation and ciliogenesis in mammalian lungs. J Cell Sci 3: 207-230

Staprans I, Dirksen ER (1974) Microtubule protein during ciliogenesis in the mouse oviduct. J Cell BioI 62: 164-174

Stockinger L, Cireli E (1965) Eine bisher unbekannte Art der Zentriolenvermehrung. Z Zellforsch 68: 733-740

Wessells N~ Evans J (1968) Ultrastructural studies of early morphogenesis and cytodifferentiation in the embryonic mammalian pancreas. Dev BioI 17 : 413-446

Wi schnitzer S (1974) Die Kernhulle: ihre Ultrastruktur und funktionelle Bedeutung. Endeavour 33: 137-142

Wynder EL, Hecht S (eds) (1976) Lung cancer. In: DICC Technical Report Series, vol 25. International Union Against Cancer, Geneva, pp95-101

Epithelial Alterations in Explant Cultures of Fetal Tracheae of Syrian Golden Hamsters

Makito Emura

Introduction

Various types of epithelial alterations have been observed in explant cultures of differentiated respiratory tissues which were treated in vitro with carcinogens (Lasnitzki 1956; Palekar et al. 1968; Crocker and Sanders 1970), infectious micro-

organisms (Gab ridge 1979), mineral dusts (Mossman et al. 1980), and other airborne particulates (Mossman and Craighead 1979). Explants taken from fetal Syrian golden hamster tracheae (13th-15th day of gestation), which were treated transplacentally with diethylnitrosamine (DEN) (see Table 1) and then cultivated for

28 Makito Emura

Table 1. List of abbreviations

l-APPN l-Acetoxypropylnitrosamine BaA Benz (aJ anthracene BAP N-Nitrosobis (2-acetoxypropyl)amine BeP Benzo(e)pyrene BHP N- Nitrosobis(2-hydroxypropyl)amine BaP Benzo(a)pyrene CHR Chrysene DBN N-Nitrosodibuthylamine DEN N- Nitrosodiethylamine; diethylnitrosamine D HPN 2,2'-Dihydroxy-di-n-propylnitrosamine DMBA 9,10-Dimethyl-l,2-benzanthracene DMDPN N- Nitrosobis(2-methylpropyl)amine DMSO Dimethyl sulfoxide DPN N-Nitrosodi-n-propylamine 2-HPPN N -Nitroso-2-hydroxypropyl-n-propylamine HEPES 4-(2-Hydroxyethyl)-l-piperazineethane sulfonic

M-2-0B MNU MOP MPN N-6-MI NM NMU 2-0PPN PAHs VEN

acid N- Nitrosomethyl(2-oxobutyl)amine Methylnitrosourea N -Nitrosomethyl(2-oxopropyl)amine N- Nitrosomethyl-n-propylamine N- Nitrosohexamethyleneimine N-Nitrosomorpholine Nitrosomethylurea N -Nitroso-2-oxopropyl-n -propyl amine Polycyclic aromatic hydrocarbons N -Nitrosovinylethylamine

4 weeks, showed changes of the same nature as those already observed in vivo. This was also true of explants treated in vitro with polycyclic aromatic hydrocarbons (PAHs). Epithelium ofthe fetal trachea is not fully differentiated at the treatment and explant stages: the cells differentiate after several weeks of cultivation. Lesions which are initiated by carcinogens at the beginning of cultivation or at the time of transplacental treatment persist within the progeny of affected cells and ultimately are manifest as morphologically discernible alterations.

Tubular Explant Culture of Fetal Tracheae

Two different methods were employed for the treatment and cultivation of fetal tracheal explants. The techniques have already been described (Emura et al. 1978, 1979; Richter-Reichhelm et al. 1982) but are summarized as follows. Randomly bred, 12-week-old Syrian golden hamster~ from the Central Proefdierenbedrijf, Zeist, The Netherlands, were maintained under standard laboratory conditions. The day of mating was regarded as day zero of gestation.

Using the first method, the pregnant females, caged individually, were anesthetized with ether and injected intraperitoneally with 200, 300, or 400 mg DEN per kilogram body wt. on day 13, 14, or 15 of gestation. DEN was dissolved in 1 ml Hanks' solution for injection. Controls received the solvent only. Fetuses were removed by cesarean section without direct contact with the mother's blood 3Yz-4h after DEN injection. The fetuses of treated and control mothers were taken out of the amniotic membranes and rinsed twice in fresh Hanks' solution. The fetal tracheae were dissected under a stereo microscope, divided into cranial and caudal portions, wrapped in chick vitellin membrane and cultivated on a membrane filter kept at the gas-medium interface under stationary conditions. Using the second method, tracheal explants of the same gestational time were treated in vitro with the following polycyclic aromatic compounds (PAHs): benzo[a]pyrene (BaP), benz[a]anthracene (BaA), benzo[e]pyrene (BeP), and chrysene (CHR) in quantities of 0.5, 1.5, and 5 mg/ml. Treatment began at 24 h after explantation and continued for 4 days. The cultivation was carried out on a rocker platform moving at 5 cycles per minute. The medium used in both methods was Eagle's minimum essential medium supplemented with fetal bovine serum to 20% during the first 4-5 days of cultivation and reduced to 5% thereafter. The total cultivation time in both experiments ranged from 3 to 9 weeks. The medium was additionally buffered with 20 mM 4-(2-hydroxyethyl)-l-piperazineethane sulfonic acid (HEPES).

Alterations After Transplacental DEN Treatment (Method 1)

Hyperplastic proliferation of basal cells (Fig. 50) and squamous metaplasia with (Fig. 51) or without cornification were frequently observed. Dysplasia and metaplasia (Fig. 52) also occurred, particularly in the highest dose group (400 mg/kg body wt.). These alterations can be seen focally in the epithelium, often in more than one site with more than one type of alteration within the same explant. Approximately two-thirds of the total treated explants showed at least one of these alterations. Furthermore, metaplastic alterations including dysplasia occur more frequently than hyperplasia (a ratio of 5: 1). No mucous cell hyperplasia can be clearly identified. Control explants treated transplacentally with a physiologi-


cally balanced solution (vehicle for DEN) produce a very well differentiated epithelium with noticeably more ciliated cells than mucous cells (Fig. 53). This is not true, however, of flattened epithelial portions opposite the membrane filter.

Fig. 50 (Above). Hyperplastic growth of cells in the ciliated epithelium of a fetal tracheal explant. Taken after 35 days of cultivation, following transplacental exposure to DEN (200 mg/kg body wt.) on the 13th day of gestation. Note stratified epithelium. Hand E, x 350

Fig.51 (Middle). Squamous metaplasia with cornification in the epithelium of a fetal tracheal explant. Taken after 42 days of cultivation, following transplacental exposure to DEN (200 mg/kg body wt.) on the 14th day of gestation.

Such flattening of the epithelium seems to occur as a result of a shortage of nutrient, the supply of which is only possible through capillary forces in the stationary type of organ culture.

The arrows indicate a vitelline membrane used for this particular organ culture technique. Hand E, x 350

Fig. 52 (Below). Dysplastic epithelium in a fetal tracheal explant after 25 days of cultivation following transplacental exposure to DEN (400 mg/kg body wt) on the 15th day of gestation. The epithelial cells show pleomorphism, cytoplasmic vacuolization, and disorientation. The arrow indicates a vitelline membrane. Hand E, x 350

30 Makito Emura

Alterations After In Vitro Treatment with P AHs (Me~hod2)

Goblet cell hyperplasia (Fig. 54) and epidermoid metaplasia (without cornification) (Fig. 55) are the two alterations most frequently induced by BaP or BaA (up to 5l!g/ml). These alterations are seen focally in the epithelium of approximately one-

Fig. 53 (Above). Epithelium in a control explant of fetal trachea excised on the 14th day of gestation and cultivated for 63 days. Ciliated and basal cells can be seen. The lumen is filled with secretion. Hand E, x 350

Fig. 54 (Middle). Mucous cell hyperplasia in the epithelium of a fetal trachea explanted on the 15th day of gestation. Treated in vitro with 5llg/ml BaP and cultivated for

third of the total explants treated with either BaP or BaA, and often occur in more than one site within the same explant. Goblet cell hyperplasia occurs more frequently than epidermoid metaplasia (a ratio of 3: 1). In the control explants treated with 0.5% dimethyl sulfoxide (DMSO) (solvent for PAHs) and the explants treated with eRR or BaP (up to 5 l!g/ ml), the frequency of squamous

28 days. Note focal proliferation of basal cells (arrows). H and E, x 350

Fig.55 (Below). Squamous metaplasia without cornification in the epithelium of a fetal trachea explanted on the 15th day of gestation. Treated in vitro with 5llg/ ml BaP and cultivated for 28 days. Hand E, x 350


metaplasia is low. Mucous cell hyperplasia cannot be clearly identified.

Conclusion

The proliferative and metaplastic alterations seen in explant cultures of fetal Syrian hamster tracheae resemble closely those so far observed in other organ culture systems (Marchok et al. 1975), animal models (Mohr et al. 1966; Gould et al. 1971; Harris et al. 1972; Becci et al. 1978), and humans (Trump et al. 1978). A similarity between the focal proliferation of basal cells seen in the BaPinduced goblet cell hyperplasia and that observed in the regenerating hamster epithelium can be seen with light microscopy (McDowell et al. 1979). Recent evidence indicates that mucous cells playa dominant role in the cell proliferation of regenerating hamster tracheal epithelium (Keenan et al. 1982). In explants treated in vitro with BaP or BaA the epithelium underwent no hyperplasia of ciliated cells or squamous metaplasia with cornification. Both of these alterations occur in explants treated transplacentally with DEN. This can be explained partly by the fact that the first method of explant cultivation promotes differentiation of ciliated cells rather than mucous cells and the second method does the reverse (Emura et al. 1978, 1979). An auto radiographic study of untreated control explants cultivated by the second method has shown that epithelial cells (indifferent cells, basal cells, and some mucus-containing cells) are labeled with 3H-thymidine (5 Meilml for 20 min of exposure) at a rate of 19% after 12 h, 14% after 24 h, and 2% after 7 days of cultivation. Although this decreasing tendency of3H-thymidine labeling during cultivation time may partly reflect the inherent mitotic activity of fetal epithelial cells (Emura and Mohr 1975), the high mitotic rates at the beginning of cultivation can be attributed to the effects of explantation and in vitro cultivation. In such mitosis-accelerating in vitro conditions the susceptibility of cells to carcinogens may well be very different from that encountered in vivo. It seems, therefore, that the difference in treatment and cultivation methods would lead to a variety of induced alterations, although the different natures of the carcinogens must also be considered. Transplacental induction of tracheal epithelial tumors with DEN is a unique system for the study of respiratory carcinogenesis, in that this particu-

lar organotypic carcinogen (DEN) can affect fetal, not fully differentiated, tracheal epithelial cells (Mohr et al. 1966). Tumor development is usually preceded by the occurrence of hyperplastic and metaplastic alterations, and there appears to exist a certain correlation between the in vivo and in vitro processes in the occurrence of such early alterations. Therefore, this in vitro system can be used for the identification of fetal cell types susceptible to DEN.

References

Becci PJ, McDowell EM, Trump BF (1978) The respiratory epithelium. IV. Histogenesis of epidermoid metaplasia and carcinoma in situ in the hamster. JNCI 61: 577-586

Crocker IT, Sanders LL (1970) Influence of vitamin A and 3,7-dimethyl-2, 6-octadienal (citral) on the effect of benzo(a)pyrene on hamster trachea in organ culture. Cancer Res 30: 1312-1318

Emura M, Mohr U (1975) Morphological studies on the development of tracheal epithelium in the Syrian golden hamster. I. Light microscopy. Z Versuchstierkd 17: 14-26

Emura M, Richter-Reichhelm HB, Mohr U (1978) Epithelial alterations in fetal tracheal explants of Syrian golden hamsters exposed to diethylnitrosamine in utero. Cancer Lett 5: 115-121

Emura M, Richter-Reichhelm HB, Emura KM, Matthei S, Mohr U (1979) Tubular explant culture of fetal Syrian golden hamster tracheae. Exp Pathol17: 196-199

Gabridge MG (1979) Hamster tracheal organ cultures as models for infection and toxicology studies. Prog Exp Tumor Res 24: 85-95

Gould VE, Wenk R, Sommers SC (1971) Ultrastructural observations on bronchial epithelial hyperplasia and squamous metaplasia. Cancer 28: 426-436

Harris CC, Sporn MB, Kaufman DG, Smith JM, Jackson FE, Saffiotti U (1972) Histogenesis of squamous metaplasia in the hamster tracheal epithelium caused by vitamin A deficiency or benzo( a}pyrene-ferric oxide. JNCI 48: 743-761

Keenan KP, Combs JW, McDowell EM (1982) Regeneration of hamster tracheal epithelium after mechanical injury. III. Large and small lesions : comparative stathmokinetic and single pulse and continuous thymidine labelling autoradiographic studies. Virchows Arch (Cell Pathol) 41: 231-252

Lasnitzki I (1956) The effect of 3-4-benz-pyrene on human foetal lung grown in vitro. Br J Cancer 10: 510-516

Marchok AC, Cone MV, Nettesheim P (1975) Induction of squamous metaplasia (vitamin A deficiency) and hypersecretory activity in tracheal organ cultures. Lab Invest 33: 451-460

McDowell EM, Becci PJ, Schiirch W, Trump BF (1979) The respiratory epithelium. VII. Epidermoid metaplasia of hamster tracheal epithelium during regeneration following mechanical injury. JNCI 62: 995-1008

Mohr U, Althoff J, Authaler A (1966) Diaplacental effect

32 Makito Emura

of the carcinogen diethylnitrosamine in the golden hamster. Cancer Res 26: 2349-2352

Mossman BT, Craighead JE (1979) Use of hamster tracheal organ cultures for assessing the cocarcinogenic effects of inorganic particulates on the respiratory epithelium. Prog Exp Tumor Res 24: 37-47

Mossman BT, Adler KB, Craighead JE (1980) Cytotoxic and proliferative changes in tracheal organ and cell cultures after exposure to mineral dusts. In: Brown RC, Gormley IP, Chamberlain M, Davis R (eds) The in vitro effects of mineral dusts. Academic, London, pp 241-250

Palekar L, Kuschner M, Laskin S (1968) The effect of.

3-methylcholanthrene on rat trachea in organ culture. Cancer Res 28: 2098-2104

Richter-Reichhelm HB, Emura M, Althoff J (1982) Scanning electron microscopical investigations on the respiratory epithelium of the Syrian golden hamster. IV. In vitro effects of dimethylsulphoxide and benzo(a)pyrene. Zentralbl Bakteriol Mikrobiol Hyg (B) 176: 269-276

Trump BF, McDowell EM, Glavin F, Barrett LA, Becci PJ, Schiirch W, Kaiser HE, Harris CC (1978) The respiratory epithelium. III. Histogenesis of epidermoid metaplasia and carcinoma in situ in the human. JNCI 61: 563-575

NEOPLASMS

Response to Carcinogens of Respiratory Epithelium, Syrian Golden Hamster (Mesocricetus Auratus)

H.-B. Richter-Reichhelm, W. Boning, and 1. Althoff

Synonyms. Papilloma; epithelial papilloma; squamous cell papilloma; epidermoid papillary tumor; polyp; adenomatous polyp; mixed polyp; polypoid tumor; papillary polyp; mucoepidermoid papillary tumor.

Gross Appearance

Early changes that may lead to neoplasia, such as focal metaplasia with loss of ciliated epithelium and epidermoid metaplasia, hyperplasia and/or papillary dysplasia, cannot be observed macroscopically, whereas progressively exophytic growing papillary tumors, whether or not they fill the airway lumen, are easily detectable under a magnifying glass or a stereo microscope (Fig. 56). In addition to their typical form and shape (sessile or

pedunculated), the tumors are soft and reddish gray, clearly distinguishable from the surrounding respiratory epithelium. Often such areas with small tumors are recognized by increased vascularization. Mter perfusion of the tissues with fixative solution, these neoplasms become a yellowish gray contrasting with the whitish adjacent epithelium.

Microscopic Features

Nonneoplastic Alterations and Precursor Lesions

Since differentiation of the pseudostratified respiratory epithelium in the Syrian golden hamster is not complete at birth, lack of ciliated cells at an early stage of the animals life may represent a

Fig.56. Papillary mucoepidermoid tumor, trachea of Syrian golden hamster, filling almost 50% of the lumen. This neoplasm was easily detectable with a dissecting microscope. SEM, x 240

34 H.-B.Richter-Reichhelm, W.Boning, and lAlthoff

physiological status rather than pathological change. In terms of surface characteristics of the neonatal epithelium, proliferative patterns prevail during the first 10 days. Less differentiated cells (probably precursors of mucous cells) and mucusproducing cells outnumber ciliated cells for the first 20 days after birth. Time of maturation of the epithelium in the trachea may vary between different topographical sites such as the pars membranacea, intercartilaginea, and cartilaginea. Differentiation of epithelium in the respiratory tract appears to be complete approximately 30 days postpartum (Richter-Reichhelm et al. 1980). If criteria for early alterations or precursor lesions are to be specific, non-compound-related degenerative and regenerative changes of the respiratory epithelium have to be recognized and considered in interpretations (McDowell et al. 1979; Althoff et al. 1981). Loss of cilia from the surface of the respiratory epithelium (simple metaplasia) develops due to acute or chronic inflammatory stimuli. Treatment with carcinogens may also cause traumatic injury at the site of application, resulting in such reactions. Thus metaplasia together with slight atypia and hyperplasia occur with conversion into a multilayered epithelium. These changes may also appear in regeneration and early neoplastic growth, which may be difficult or impossible to distinguish morphologically. Metaplastic and hyperplastic changes in the respiratory epithelium may occur focally, with a marked, well-circumscribed border, or multifocally, less defined and/or disseminated. Simple metaplasia of the ciliated, pseudostratified epithelium lining the airways is easily recognized by loss of cilia and flattening of the epithelial cells. In the cuboidal unilayered epithelium, mucous granules and cilia are only occasionally found; cell nuclei are round or oval and mitotic activity is usually increased.

Hyperplasia. The lesions may grow into a multilayered pattern by an increase in the number of basal cells (so-called basal cell hyperplasia). Less differentiated cells constitute the intermediate layer between the basal cells and surface lining. These cells may be either indifferent or highly differentiated (e.g., mucous cell hyperplasia).

Squamous Metaplasia. Surface cells are flattened and have horizontally orientated nuclei (parallel to the basal lamina); they may be nonkeratinized or contain keratohyaline granules. In these lesions, changes such as acanthosis, parakeratosis,

hyperkeratosis, and dyskeratosis may be associated with squamous metaplasia. Desquamation of surface cells is often observed.

Dysplasia; Carcinoma In Situ. Circumscribed areas with a slightly elevated, dome-shaped surface are composed of cells with irregular shapes and sizes that proliferate toward the lumen (Fig. 57). In profile, they are irregular in shape and folded. The hyperplastic lesion consists of one or two layers of basal cells (term used in light microscopy) covered by four or five layers of less differentiated (intermediate) cells, sometimes with increased mitotic activity and atypism. Loss of orientation and organization of cells is evident. In cross sections of the epithelium, varying cell shapes are seen; folds appear as papillary projections into the lumen. The uppermost luminal layer is made up either of flat squamous or mucus-producing cells. Single, ciliated cells occur occasionally. The pleomorphism of the cells and the irregularity in cell surface patterns are indicative of dysplasia (Fig. 58) (carcinoma in situ) (Becci et al. 1978).

Papillary Tumors

Exophytic papillary neoplastic alterations of the respiratory epithelium have largely the same cell surface pattern (i. e., epidermoid, less differentiated mucus-producing, and ciliated cells) as those seen in dysplasia (Fig. 59). The epithelial portion of the tumors is supported by a delicate vascularized mesenchymal stroma (Fig. 60). Secondary inflammatory changes are frequent. The basallamina is seen in all branches of the papillary exophytic growth and is generally not disrupted. If the epithelium of such a papillary neoplasm is composed solely of squamous cells, it may be regarded as an epidermoid papillary tumor. Sections reveal a flattened layer of cells with horizontally oriented nuclei (parallel to the basal membrane). Keratin may be formed in some instances. A papillary mucoepidermoid tumor consisting of both mucus-producing and epidermoid cells (Fig. 61) is comparatively easily distinguished by applying Kreyberg stain (mucus = green, keratin = red) (Kreyberg 1967). In addition to the presence of differentiated mucus-producing cells at the tumor surface and intraepithelial cysts, cells containing mucous granules are found in the intermediate zone. Mitoses are seen in all cell layers, although they are not very frequent. Subepithelial

Response to Carcinogens of Respiratory Epithelium, Syrian Golden Hamster 35

Fig.57 (Above). Circumscribed area of trachea consisting of cell surfaces varying in shape and form indicating hyperplasia and dysplasia. Note folds and slight projection into the tracheal lumen. SEM, x 320

mucous glands may also undergo metaplasia and proliferative changes, a feature which could be falsely interpreted as invasive growth. In addition, papillary tumors may originate in the cystic ducts of the subepithelial glands.

Fig.58 (Below). Dysplastic alteration (carcinoma in situ) of the respiratory epithelium, trachea. Note multilayered atypical cell arrangement with vacuoles and epidermoid cells in the uppermost layer. Semithin section, toluidine blue, x 350

Ultrastructure

Nonneoplastic and Precursor Lesions

Simple Metaplasia. In simple metaplasia, cells are marked by the loss of cilia and intracytoplasmic ciliary bodies. Fewer cilia than normal may occur in some cells and occasionally cells with cytoplasmic protrusions may contain cilia. In cross sec-

36 H.-B. Richter-Reichhelm, W. Boning, and 1. Althoff

Fig.59 (Above). Early mucoepidermoid papillary tumor, trachea, with intraepithelial cysts and focal squamous metaplasia. Note the delicate vascularized connective tissue stalk (S). In some areas under the basal lamina early tumor growth seems to interrupt the basement membrane (arrows). Semithin section, toluidine blue, x 150

tions of these cilia, micro filamentous patterns appear to be normal, with nine doublets around the periphery and two in the center. In some instances, degenerative changes such as swelling of mitochondria and multivesiculated and laminated bodies (secondary Iysosomes) may be found. The luminal cell surface bears short and numerous microvilli; cell borders are clearly distinguish-

Fig.60 (Lower left). Mucoepidermoid papillary tumor of the respiratory epithelium filling the tracheal lumen. Note delicate mesenchymal stroma with capillaries (S). Hand E, x 80

Fig.61 (Lower right). Epithelial cells, trachea, containing mucus (M) (green in Kreyberg stain). At the apex (a), flattened cells with reddish cytoplasm indicate nonkeratinized squamous cells. Kreyberg stain, x 440

able by the slightly elevated cell membrane and increased density of cytoplasmic protrusions. Generally, all cells have a similar shape and size and nuclei are oval and uniform.

Squamous Metaplasia. In epidermoid metaplasia of the respiratory epithelium, the alignment of cellular and nuclear axes parallel to the basal


membrane is easily recognizable. Many cells in the now multilayered epithelium contain bundles of tonofilaments. Keratin granules are not always detectable. The number of desmosomes has largely increased and interdigitations of cell membranes are pronounced. In some areas of epidermoid metaplasia, nuclei are flat and deeply invaginated.

Hyperplasia. In a thick epithelium, with general loss of cilia at the luminal cell surface, ciliogenesis may be disturbed as cells appear to produce premature intracytoplasmic cilia. These cilia may be found even at infranuclear sites close to the basal epithelial layers. In other areas, less differentiated and highly differentiated mucous cells may compose the majority of cells in focal lesions (mucous cell hyperplasia). These cells have highly developed and conspicuous endoplasmic reticulum, as well as Golgi complexes. Large mucous granules may not be present in all cells.

Dysplasia. Ultrastructural features of epithelial cells in sites of cellular atypia include empty vacuoles, intraepithelial cysts filled with mucus, and occasionally structurally intact cilia. These features lead to the diagnosis of dysplasia or carcinoma in situ. Varying degrees of cellular differentiation form the epithelial pattern, particularly of the surface structure. On some occasions, dense core granules, as seen in neurosecretory cells or

"amine precursor uptake and decarboxylation" (APUD) cells, are formed. Small areas can be seen which contain squamous cells at the irregularly folded luminal surface of the epithelium. Cell nuclei vary in size and form; they often contain deep invaginations and atypical mitotic figures.

Papillary Tumors

The structure, seen by surface electron microscopy, of papillary tumors arising from the respiratory epithelial lining may have varying patterns. If the surface of the tumor cells is covered with patterns arranged in microfolds and microridges, characteristic of nonkeratinized squamous cells, the diagnosis of epidermoid tumor can be made. Frequent mitoses support the proliferative nature of this exophytic neoplastic growth. In some instances desquamation of flat surface cells may be prominent, exposing the surface of underlying less differentiated cells. Some papillary tumors have the features of polymorphism similar to that seen in dysplasia. In addition to the less differentiated areas consisting of cells with varying numbers of microvilli, groups of well-differentiated cells with cilia and mucus are found (Fig. 62). Ciliogenesis and mucogenesis may not be restricted to the surface layer (Figs. 63 and 64). In other parts of the same tumor, numerous desmosomes may be found, and some cells

Fig. 62. Early papillary tumor, trachea. The surface of the mass consists of squamous mucus-producing and ciliated cells. SEM, x 420


contain increased numbers of bundled filaments. Cell boundaries are digitated, nuclei are flat and invaginated (Fig. 65). Keratohyaline granules may be found occasionally and sometimes cells situated in the intermediate layer contain neurosecretory granules. Intracytoplasmic vacuoles and intraepithelial cysts are frequent. Usually, the basal lamina is intact throughout the entire tumor stalk. In these papillary tumors, mitotic figures may be greater in number than in dysplasia.

Differential Diagnosis

Papillary tumors of the surface respiratory epithelium (nasal cavities, larynx, trachea, bronchi) are classified according to their epithelial component, i. e. neoplasms with squamous epithelium on their surface (nonkeratinizing or keratinizing) are classified as epidermoid (or squamous) papillary tumors. Exophytic neoplasms which contain goblet cells, an occasional ciliated cell, and squamous cells should be named mucoepidermoid papillary tumors. These tumors may also be made up of areas of less differentiated cells. Their exophytic growth is usually noninvasive. When infiltration occurs, the neoplasms gain characteristics of epidermoid or mucoepidermoid carcinomas. Routine histology (hematoxylin-eosin or toluidine blue stain) may render sufficient information in terms of neoplastic growth, but only special stains such as PAS reaction, alcian blue, and/or Kreyberg's method (Kreyberg 1967) allow the cytological identification of cell status necessary for differential diagnosis (Herrold 1964a; Herrold and Dunham 1963; Dontenwill and Mohr 1961, 1962; Mohr 1970). Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) preparations may provide evidence of histiogenesis, especially when the tissue is less differentiated.

<l Fig. 63 (Above). Mucus-producing cell from a mucoepidermoid respiratory tumor, bronchus, hamster. Note the Golgi complex (G) and mucous granules (M). x 47500

Fig.64 (Middle). Mucoepidermoid respiratory tumor, bronchus, revealing multiple ciliary bodies (C) in a cell attached to the basal lamina. TEM, x 9500

Fig.65 (Below). Portion of a respiratory mucoepidermoid tumor, bronchus, in a flattened cell layer. Nuclei are digitated (N), cytoplasm contains bundled tonofilaments (E). Note abundant desmosomes (D). TEM, x 6900

Biologic Features

Papillary tumors of the larynx, trachea, and bronchi develop as single or multiple neoplasms. They often originate in the epithelium of the membranaceous portion if cartilage partially composes the airway. The tumors sometimes occlude the lumen, causing death by suffocation. Neoplasms that partially close the airway lumen can cause irregular breathing. Distal portions of the respiratory tract may undergo atelectases and emphysema, and infections of the lung enhanced by hampered clearance and defense mechanisms often occur. Papillary tumors arising in the respiratory epithelium of the larynx, trachea, and bronchi may be tom from their site of origin and implanted into the lung after aspiration (Sellakumar et al. 1976). This could easily be the case if the neoplasms are pedunculated and treatment by intralaryngeal and intratracheal instillation techniques is continued after tumors have already occurred. In general, metastasis of these papillary tumors of the epithelial lining of the respiratory tract do not occur frequently. Only occasionally is invasive growth found in the mediastinum (Herrold 1964b; Althoff et al. 1977). Papillary tumors also arise in the ducts of subepithelial glands. Since the epithelium of these glands also undergoes metaplasia, difficulties may occur in diagnosing true invasive growth. They may be malignant, since they are transplantable into other tissues. The respiratory epithelium of the Syrian golden hamster is highly sensitive to the carcinogenic effects of systemically acting compounds as well as to locally applied agents. The early and advanced stages of papillary tumors described here have developed after exposure to a carcinogen (nitrosamines, polycyclic aromatic hydrocarbons). Relatively few tumors of the larynx, trachea, and bronchi have been reported in untreated hamsters (Pour et al. 1976a; Homburger et al. 1983).

Comparison with Other Species

Compared with other laboratory animals, spontaneous pathological alterations and neoplastic changes of the respiratory epithelium in Syrian golden hamsters are infrequent (Nettesheim 1972; Pour et al. 1976a; Mohr and Richter-Reichhelm 1982). Nonneoplastic alterations, caused mainly by infections, are found in all species. Due to specific anatomic conditions, sites where metaplasia occurs more frequently have been described in

40 H.-B. Richter-Reichhelm, W. Boning, and 1. Althoff

man (Auerbach et al. 1962), pigs, dogs, and rabbits (Wang et al. 1972). Squamous metaplasia is very common at ductal openings of subepithelial glands and bifurcations of the tracheobronchial tree. This phenomenon has been attributed to the heavy deposition of inhaled particles at these particular sites, resulting in intense and prolonged alterations of the function of the mucociliary clearance system. If the results of experimental respiratory tract carcinogensis are to be interpreted accurately, the rat as an experimental model should be considered with caution. Chronic endemic bronchitis (see p. 211), which results in severe regenerative and dysplastic alterations progressing to overt bronchiectasis, is commonly found in many strains (Pour et al. 1976b). Very little is known about spontaneous lesions in the main airways of mice. Certain differences exist between tumors of the upper respiratory system (larynx, trachea, bronchi) in hamsters and humans. In man papillary tumors of the larynx are more frequent than those of the trachea, but in hamsters tracheal neoplasms predominate. In the hamster, this reflects the effect of carcinogenesis on a specific anatomic localization. Tumors which mainly occur in young human patients may be multiple or even diffuse, involving the entire tracheobronchial tree. For a long time, single papillary tumors were described as developing predominantly in the stem bronchi and carina, and also occasionally in the main and segmental bronchi (Liebow 1952). From the histogenetic point of view, the induced epithelial changes described in the respiratory tract of the Syrian golden hamster are quite comparable to findings in human bronchi (McDowell et al. 1982; Melamed and Zaman 1982). Other hamster species, such as the European hamster (Cricetus cricetus L) and the Chinese hamster ( Cricetulus griseus M. E.) have been investigated in studies of respiratory tract carcinogenesis. The Syrian golden hamster (Mesocricetus auratus W) should be regarded as the most appropriate model, partly because of the knowledge of the physiology and pathology of lesions of this species (Mohr and Reznik 1982).

References Althoff J, Grandjean C, Russell L, Pour P (1977) Vinyl

ethylnitrosamine: potent respiratory carcinogen in Syrian hamsters. JNCI 58: 439-442

Althoff J, Richter-Reichhelm HB, Green U, Kracke D (1981) Scanning electron microscopical investigations on the respiratory epithelium of the Syrian golden hamster. III Regeneration after traumatic injury. Zentralbl Bakteriol (B) 174: 249-259

Auerbach 0, Stout AP, Hammond EC, Garfinkel L(1962) Bronchial epithelium in former smokers. N Engl J Med 267:119-125

Becci PJ, McDowell EM, Trump BF (1978) The respiratory epithelium. IV. Histogenesis of epidermoid metaplasia and carcinoma in situ in the hamster. JNCI 61: 577-586

Dontenwill W, Mohr U (1961) Carcinome des Respirationstractus nach Behandlung von Goldhamstern mit Diathylnitrosamine. Z Krebsforsch 64: 305-312

Dontenwill W, Mohr U (1962) Vergleichende Untersuchungen an metaplastischen und malignen Epithelveranderungen des Respirationstraktes im Tierexperiment. Z Krebsforsch 65: 168-170

Herrold KM (1964a) Epithelial papillomas of the nasal cavity. Arch Pathol78: 189-195

Herrold KM (1964b) Effect of route of administration on the carcinogenic action of diethylnitrosamine (N-nitrosodiethylamine). Br J Cancer 18: 763-767

Herrold KM, Dunham U (1963) Induction of tumors in the Syrian hamster with diethylnitrosamine (N-nitrosodiethylamine). Cancer Res 23 :773-777

Homburger F, van Dongen CG, Adams R, Soto E (1983) Standardizing Syrian hamsters for toxicology. In: Homburger F (ed) Safety evaluation and regulation of chemicals. Karger, Basel, pp 225-232

Kreyberg L (1967) International histological classification of tumours. No 1: Histological typing of lung tumours. WHO, Geneva

Liebow AA (1952) Tumors of the lower respiratory tract. In: Atlas of tumor pathology, 1st series, Fasicle 17. Armed Forces Institute of Pathology, sect 5, Washington DC

McDowell EM, Becci PJ, Schurch W, Trump BF (1979) The respiratory epithelium. VII. Epidermoid metaplasia of hamster tracheal epithelium during regeneration following mechanical injury. JNCI 62: 995-1008

McDowell EM, Harris CC, Trump BF (1982) Histogenesis and morphogenesis of bronchial neoplasms. In: Shimosato Y, Melamed MR, Nettesheim P (eds) Morphogenesis oflung cancer, vol 1. CRC, Boca Raton, pp 1-36

Melamed MR, Zaman MB (1982) Pathogenesis of epidermoid carcinoma of the lung. In: Shimosato Y, Melamed MR, Nettesheim P (eds) Morphogenesis of lung cancer, vol 1. CRC, Boca Raton, pp 37 -64

Mohr U (1970) Effects of diethylnitrosamine in the respiratory system of Syrian golden hamsters. In: Nettesheim P, Hanna MG, Deatherage JW (eds) Morphology of experimental respiratory carcinogensis, AEC Symposium Series No 21. USAEC, Division of Technical Information Extension, Oak Ridge, pp 255-265

Mohr U, Reznik G (1982) Three hamster species as models in cancer research. In: Turusov VS (ed) Pathology oftumours in laboratory animals, vol III. Tumours of the hamster. IARC Sci Pub134: 437-442

Mohr U, Richter-Reichhelm HB (1982) The impact of spontaneous pathology on the quality of animals for toxicological studies. In: Bartosek I, Guaitani A, Pacei E (eds) Animals in toxicological research. Raven, New York, pp 65-70

Nettesheim P (1972) Respiratory carcinogenesis studies with the Syrian golden hamster: a review. Prog Exp Tumor Res 16: 185-200

Pour P, Mohr U, Cardesa A, Althoff J, Kmoch N (1976 a) Spontaneous tumors and common diseases in two colonies of Syrian hamsters. II. Respiratory tract and digestive system. JNCI 56: 937-948

Pour P, Stanton MF, Kuschner M, Laskin S, Shabad LM (1976 b) Tumours of the respiratory tract. In: Turusov VS

Polypoid Adenoma, Nasal Mucosa, Rat 41

(ed) Pathology of tumours in laboratory animals, vol 1. Tumours of the rat, part 2. IARC Sci Pub16: pp 1-61

Richter-Reichhelm HB, Emura M, Althoff J (1980) Scanning electron microscopical investigations on the respiratory epithelium of the Syrian golden hamster. I. Postnatal differentiation. Zentralbl Bakteriol (B) 171: 424-432

Sellakumar A, Stenback F, Rowland J (1976) Effects of different dusts on respiratory carcinogenesis in hamsters induced by benzo( a )pyrene and diethylnitrosamine. Eur J Cancer 12: 313-319

Wang N-S, Huang S-N, Thurlbeck WM (1972) Squamous metaplasia of the opening of bronchial glands. Am J Pathol67: 571-582

Polypoid Adenoma, Nasal Mucosa, Rat

William D. Kerns

Synonyms. Adenomatous polyp; papillary adenoma; adenoma; papilloma.

Gross Appearance

Polypoid adenomas are usually observed in the most anterior part of the nasal cavity (Kerns et al. 1983a, b). They may vary in size from small microscopic nodules to masses large enough to protrude from the nares and may cause dyspnea. While the adenomas are noninvasive, larger ones may cause obstruction of the nasolacrimal duct or

Fig.66. Coronal section of nasal cavity from a rat following exposure to formaldehyde. A small polypoid adenoma (arrows) has arisen on the lateral side of the maxilloturbinate. ND, nasolacrimal duct; N, nasoturbinate; M, maxilloturbinate; V, vomeronasal organ

induce pressure atrophy of adjacent structures. In formalin-fixed decalcified coronal sections, the tumor appears as a solid, whitish gray mass (Fig. 66).


Polypoid adenomas may be sessile (Fig. 67) or pedunculated (Fig.68). They arise from the mucosa of the nasoturbinates, maxilloturbinates, or lateral wall of the anterior nasal cavity. The lamina propria contains few inflammatory cells and little

42 William D. Kerns

supporting connective tissue, but is well vascularized. Nonciliated tumor cells of the adenomas vary morphologically from cuboidal to low columnar epithelium; they form solid sheets or microcysts. In hematoxylin and eosin preparations, the cells are recognized by basophilic cytoplasm and centrally located nuclei. The cysts contain PAS-positive material, sloughed epithelium, and inflammatory cells (Fig. 67). Adenomas appear to originate as hyperplastic, exophytic, mucosal nodules. Origin from the submucosa seems unlikely, since tumors frequently arise in areas that are devoid of submucosal glands and the tumor cells have light and electron microscopic features of respiratory epithelium (Monteiro-Riviere and Popp 1984). Morphological characteristics of larger tumors suggest that, with continued growth, the surface epithelium invaginates into the tumor mass, thus forming microcysts (Fig. 69).

Ultrastructure

Adenomas consist of nonciliated electron-dense (dark) and electron-lucent (light) cuboidal and low columnar epithelial cells (Fig.70). Dark cells can be recognized by their short, nonbranching

microvilli, centrally located convoluted nuclei, small apical vacuoles, basal clusters of mitochondria, and electron-dense cytoplasm (Fig. 71). Light cells are also characterized by centrally located nuclei, but with fewer indentations of the nuclear membrane. Light cell cytoplasm is electron lucent and contains small apical vacuoles and randomly distributed mitochondria. Most but not all light cells have short, nonbranching microvilli (Fig. 72). The cell membranes of both types of cells interdigitate extensively, and most cells have a single desmosome in their apical junctional zone. Light and dark cells appear to have equal complements of rough endoplasmic reticulum and polyribosomes. Ultrastructurally, the cells comprising polypoid adenomas have the characteristics of respiratory epithelium (Monteiro-Riviere and Popp 1984).


Polypoid adenomas should pose few diagnostic problems. In some cases, it may be difficult to determine whether the cell of origin is of respiratory epithelium or submucosal glandular epithelium. Klaassen et al. (1982) have reported that the submucosal glandular epithelium is characterized

Fig. 67. A sessile polypoid adenoma from the nose of a rat folowing exposure to formaldehyde. Microcysts contain PASpositive material. Hand E, x 230 (reduced by 15%)

Fig.68 (Above). A pedunculated polypoid adenoma of the mucosa, maxilloturbinate of a rat after exposure to formaldehyde. Hand E, x 180


Fig.69 (Below). A polypoid adenoma of the nasoturbinate of a rat after exposure to formaldehyde . Apparent invagination (arrows) of the surface mucosa. Hand E. x 360

44 William D. Kerns

Fig. 70. Light (L) and dark (D) cells in a polypoid nasal adenoma in a rat after exposure to formaldehyde. A small cyst filled with neutrophils and debris is present. TEM, x 2500 (reduced by 15%)

Fig.71. A nonciliated dark cell from an adenoma, recognized by its apical vacuoles, basal clusters of mitochondria, and electron-dense cytoplasm. Note elaborate interdigitation of the cell membrane (M), characteristic of nasal respiratory epithelium. TEM, x 8000 (reduced by 15%)

Fig. 72. A non ciliated light cell from an adenoma, recognized by its small apical vacuoles, randomly distributed mitochondria, and electron-lucent cytoplasm. Both light and dark cells usually have short, nonbranching microvilli (M). TEM, x 8000 (reduced by 15%)

ultrastructurally by apical secretory granules (0.5-1.0 ~m) of varying electron density and welldeveloped rough endoplasmic reticulum. Ultrastructural examination and/ or immunohistochemical reactions for keratin (aKl antigen) may be helpful in identifying periacinar myoepithelial cells. Myoepithelial cells and large secretory granules would normally be found only in adenomas originating from the submucosal glands (Nathrath et al. 1982; Klaassen et al. 1982).

Biologic Features

A polypoid adenoma has been reported, as a spontaneous lesion, in a male Fischer 344 rat (Kerns et al. 1983 b), and this tumor has been observed in control and treated rats from toxicity studies of a variety of chemical compounds (Table 2; Stinson 1983). No evidence is available to suggest that polypoid adenomas progress to adenocarcinomas of the nasal cavity in the rat. It has been reported (Takano et al. 1982) that focal nodular hyperplasia (inverted papilloma) is much more important in the


pathogenesis of nasal adenocarcinoma than are polypoid adenomas (exophytic papilloma).


In humans, epithelial papillomas (also termed nasal papillomatosis, squamous papilloma, squamous papillary epithelioma, Schneiderian papilloma, cylindric papilloma, or transitional cell papilloma), not adenomas, are the most common benign neoplasms of the nasal cavity. Even so, they are relatively rare lesions, being only 1125th as frequent as the more common inflammatory nasal polyp (Hyams 1971; Lasser et al. 1976; Ridolfi et al. 1977; Snyder and Perzin 1972). Morphologically similar neoplasms, identified as microcystic papillary adenomas, have been reported in humans, and they represent 1.6% of all tumors of the nose and sinuses (Friedmann and Osborn 1982). Malignant transformation has not been encountered. Nasal adenomas have been reported in dogs and cats (Madewell et al. 1976), but morphological descriptions were not provided. Transmissible ovine

46 William D. Kerns

Table 2. Polypoid adenomas observed in the nasal cavity of the rat

Chemical Strain Male"

Formaldehyde F344 0.0 ppm 1/118 2.0 ppm 4/118 5.6 ppm 6/119

14.3 ppm 4/117

Phenacetin S-D 1.25% 3/22 2.5% 0127

p-Cresidine F344 0.5% 1/48 1.0% 2/45

1.4-Dinitrosopiperazine F344 0.01% 81/125

Hexamethylphosphoramide S-D 100ppb 11200b

Nitrosaminobutanone F344 11.7 mg/ day for 60 days (SC) 4/12c

2,6-Xylidine S-D 1000 ppm 2/56 3000 ppm 10/56

Ethyl acrylate F344 Oppm 1/59 o ppm 0/62

25 ppm 0/77 75 ppm 0178

225 ppm 1170

SD. Sprague-Dawley; ND. not determined a No. of tumors/no. of nasal cavities evaluated b Sex not reported c No. of tumors represents adenomas and papillomas

adenomas, adenopapillomas, and adenocarcinomas have been reported (Njoku et al. 1978; Yonemichi et al. 1978), but are morphologically distinct from polypoid adenomas of the rat. Adenomas have also been reported in mice (Reznik et al. 1980), gerbils (Cardesa et al. 1976), and hamsters (Feron et al. 1982). Adequate morphological descriptions and photomicrographs were not provided and it is not certain if these tumors are similar to those of the rat.

Female" Study type Reference

Inhalation Kerns et al. 0/114 1983a, b 4/118 0/116 1/115

Feeding Isaka et al. 3125 1979 1127

Feeding Reznik et al. 1/46 1981 6/47

Drinking Takano et al. ND water 1982

Inhalation Lee and Trochimowicz 1982

Injection Hecht et al. 6/12c 1980

0/56 Feeding Kornreich and 6/56 Montgomery

1984

1/60 Inhalation 1. Young personal 0/60 communication 1175 1175 0171

References

Cardesa A, Pour P, Haas H, Althoff J, Mohr U (1976) Histogenesis of tumors from the nasal cavities induced by diethylnitrosamine. Cancer 37: 346-355

Feron VJ, Kruysse A, Woutersen RA (1982) Respiratory tract tumours in hamsters exposed to acetaldehyde vapour alone or simultaneously to benzo(a)pyrene or diethylnitrosamine. Eur J Cancer Clin Oncol18: 13-31

Friedmann I, Osborn DA (1982) Tumours of the mucosal glands. In: Friedmann I, Osborn DA (eds) Pathology of granulomas and neoplasms of the nose and paranasal sinuses. Churchill Livingstone, New York, 133-161

Hecht SS, Chen C-HB, Ohmori T, Hoffmann D (1980) Comparative carcinogeniticy in F344 rats of the tobacco-specific nitrosamines, N' -nitrosonornicotine and 4-(N-methyl-N-nitrosamino )-1-(3-pyridyl)-1-butanone. Cancer Res 40: 298-302

Hyams VJ (1971) Papillomas of the nasal cavity and paranasal sinuses. A clinicopathological study of 315 cases. Ann Otol Rhinol Laryngol80: 192-206

Isaka H, Yoshii H, Otsuji A, Koike M, Nagai Y, Koura M, Sugiyasu K, Kanabayashi T (1979)Tumors of SpragueDawley rats induced by long-term feeding of phenacetin. Gan 70: 29-36

Kerns WD, Donofrio DJ, Pavkov KL (1983 a) The chronic effects of formaldehyde inhalation in rats and mice. A preliminary report. In: Gibson JE (ed) Formaldehyde toxicity. Hemisphere, New York, pp 111-113

Kerns WD, Pavkov KL, Donofrio Dl, Gral1a El, Swenberg lA (1983 b) Carcinogenicity of formaldehyde in rats and mice after long-term inhalation exposure. Cancer Res 43: 4382-4392

Klaassen ABM, lap PHK, Kuijpers W (1982) Ultrastructural aspects of the nasal glands in the rat. Anat Anz 151: 455-466

Kornreich M, Montgomery CA (1984) Technical report on the carcinogenesis bioassay of 2,6-xylidine (2,6-dimethylanaline). Charles River C-D rats (diet study). CAS 87-62-7

Lasser A, Rothfeld PR, Shapiro RS (1976) Epithelial papilloma and squamous cel1 carcinoma of the nasal cavity and paranasal sinuses: a clinicopathological study. Cancer 38: 2503-2510

Lee KP, Trochimowicz HI (1982) Induction of nasal tumors in rats exposed to hexamethylphosphoramide by inhalation. INCI 68: 157-171

Madewel1 BR, Priester WA, Gillette EL, Snyder SP (1976) Neoplasms of the nasal passages and paranasal sinuses in domesticated animals as reported by 13 veterinary col1eges. Am 1 Vet Res 37: 851-856

Monteiro-Riviere N, Popp lA (1984) Ultrastructural characterization of the nasal respiratory epithelium in the rat. Am 1 Anat 169: 31-43

Nathrath WB, Wilson PD, Trejdosiewicz LK (1982) Immunohistochemical localisation of keratin and luminal

Neoplasms, Mucosa, Ethmoid Turbinates, Rat 47

epithelial antigen in myoepithelial and luminal epithelial cells of human mammary and salivary gland tumours. Pathol Res Pract 175: 279-288

Njoku CO, Shannon D, Chineme CN, Bida SA (1978) Ovine nasal adenopapilloma: incidence and clinicopathologic studies. Am 1 Vet Res 39: 1850-1852

Reznik G, Reznik-Schiil1er HM, Hayden DW, Russfield A, Murthy ASK (1981) Morphology of nasal cavity neoplasms in F344 rats after chronic feeding of p-cresidine, an intermediate of dyes and pigments. Anticancer Res 1: 279-286

Reznik G, Ul1and B, Stinson SF, Ward 1M (1980) Morphology and sex-dependent manifestation of nasal tumors in B6C3F1 mice after chronic inhalation of 1,2-dibromo-3-chloropropane. 1 Cancer Res Clin Oncol 98: 75-83

Ridolfi RL, Lieberman PH, Erlandson RA, Moore OS (1977) Schneiderian papillomas: a clinicopathologic study of 30 cases. Am 1 Surg Pathol1 : 43-53

Snyder RN, Perzin KH (1972) Papillomatosis of nasal cavity and paranasal sinuses (inverted papilloma, squamous papilloma). A clinicopathologic study. Cancer 30: 668-690

Stinson SF (1983) Nasal cavity cancer in laboratory animal bioassays of environmental compounds. In: Reznik G, Stinson SF (eds) Nasal tumors in animals and man, vol III. Experimental carcinogenesis. CRC, Boca Raton, chap 7

Takano T, Shirai T, Ogiso T, Tsuda H, Baba S, Ito N (1982) Sequential changes in tumor development induced by 1,4-dinitrosopiperazine in the nasal caity of F344 rats. Cancer Res 42: 4236-4240

Yonemichi H. Ohgi T, Fujimoto Y, Okada K, Onuma M, Mikami T (1978) Intranasal tumor of the ethmoid olfactory mucosa in sheep. Am 1 Vet Res 39: 1599-1606

Neoplasms, Mucosa, Ethmoid Turbinates, Rat

Sherman F. Stinson and Hildegard M. Reznik-Schuller

Synonyms. Adenocarcinoma; squamous cell carcinoma; olfactory neuroblastoma, esthesioneuroblastoma.

Gross Appearance

Neoplasms of the ethmoid regions of the nasal cavities of rats appear, in advanced cases, as large masses exhibiting both exophytic and endophytic patterns of growth. One or both sides of the posterior nasal cavities may be completely filled by the tumor, resulting in displacement, erosion, and destruction of the septum, turbinals, and other sur-

rounding osseous and cartilaginous structures. Extension through the cribriform plate into the olfactory lobes of the brain can frequently be visualized grossly upon longitudinal or coronal sectioning.


Three major types of neoplasms have been reported to occur in the ethmoid regions of the nasal cavities of rats: adenocarcinomas, squamous cell carcinomas, and olfactory neuroblastomas. The adenocarcinomas are usually poorly differentiat-

48 Shennan F. Stinson and Hildegard M. Reznik-Schuller

Fig.73. Poorly differentiated adenocarcinoma of the ethmoturbinal regional of a F344 rat fed a diet containing p-cresidine for 2 years. Disorganized masses of cells with round to oval nuclei and little cytoplasm are seen. Hand E, x 130

ed and consist of masses of anaplastic cells having scant cytoplasm and large round to oval hyperchromatic nuclei demonstrating considerable pleomorphism (Fig. 73). Bizarre mitotic figures are common (Fig.74). The pattern of differentiation varies in different parts of the neoplasms - some areas contain relatively well-differentiated cells and arrangement, while most areas appear less well differentiated with a lack of cellular organization and orientation (Fig. 75). Some lesions have a higher proportion of well-differentiated areas with glandular architecture. Fine fibrovascular septa course through the tumors, dividing them into lobules. Most adenocarcinomas contain areas with formation of rosettes, pseudorosettes or small acinar structures. These rosettes are formed by tumor cells, usually poorly differentiated or anaplastic, that are oriented around a centrallumen or blood vessel (Figs. 73 and 74). Areas of squamous differentiation are also found within these neoplasms (Fig. 76). Poorly differentiated adenocarcinomas invade into and through the bones surrounding the posterior nasal cavities

Fig.74. Higher magnification of the poorly differentiated adenocarcinoma in Fig. 73. Considerable pleomorphism and a high mitotic rate are in evidence. Organization of cells into rosette structures is seen. Hand E, x 330

(Fig. 77) and through the cribriform plate into the olfactory lobes of the brain (Fig. 78). Areas of invasion have architectural and cytological characteristics typical of the primary tumor. Metastases occur frequently, most commonly to the cerebrum, regional lymph nodes, and lung. Squamous cell carcinomas are usually poorly differentiated and exhibit the same histological features as squamous cell carcinomas found in the anterior regions of the nasal cavities. The histologic features are discussed in detail on page 54. As with adenocarcinomas, squamous cell carcinomas of the olfactory region frequently invade the brain, but metastases are less common. Diagnostic microscopic criteria for olfactory neuroblastoma have been defined in detail (Obert et al. 1960) and comprise the following: plexiform intercellular fibrils, poorly defined almost nonexistent cytoplasm, round to oval nuclei, distinct sharply defined nuclear chromatin, compartmentalization of sheets of neoplastic cells into lobules by fibrovascular septae, rosettes, and pseudorosettes. These features are very similar to those ob-

Fig. 75. Adenocarcinoma induced in the ethmoid region of a F344 rat with N-nitrosomethylpiperazine. Widely varying degrees of differentiation are present. Hand E, x 200

served in poorly differentiated adenocarcinomas of the ethmoid region, which undoubtedly has led to diagnostic errors in some cases. For example, electron microscopy and reexamination of histological material from a study in which p-cresidine was reported to induce esthesioneuroblastomas revealed no features of neurogenic origin in the tumors. Origin from basal cells of the olfactory epithelium was suggested and the diagnosis of adenocarcinoma was made (Reznik et al. 1981). Diagnosis of olfactory neuroblastoma should rely on the presence of neurogenic features (best demonstrated by electron microscopy), such as axons, neurofibrils, neurotubules, and dense-cored secretory granules. Most studies in rats where olfactory neuroblastomas have been reported have not included this verification, and therefore such diagnoses are in question.

Ultrastructure

Very few studies of tumors in the ethmoid region of the rat nasal cavities have included electron microscopic examination. One neoplasm observed ultrastructurally was induced in a F344 rat


Fig. 76. Squamous differentiation within an adenocarcinoma induced in the ethmoid region of a F344 rat with Nnitrosomethylpiperazine. Hand E, x 300

by chronic inhalation of the soil fumigant nematocide 1,2-dibromo-3-chloropropane (3 ppm, 6 h/ day, 5 days/week). The tumor had infiltrated the brain and nasal bones at the time the animal was killed. By light microscopy, the tumor was classified as poorly differentiated adenocarcinoma. Electron microscopy (Reznik-SchUller, unpublished results) revealed junctional complexes which linked the tumor cells together. The cytoplasm contained numerous ribosomes and polyribosomes, while rough endoplasmic reticulum and mitochondria were scanty (Fig. 79 and 80). A few of the neoplastic cells possessed long, slender microvilli at their luminal surfaces (Fig. 80). However, in no cell were dense-cored granules, neurotubules or axon-like extensions detected. It was concluded that this tumor was derived from basal cells with some differentiation toward sustentacular features.


The major differential diagnostic problem is between poorly differentiated adenocarcinoma and olfactory neuroblastoma (esthesioneuroblasto-

50 Sherman F. Stinson and Hildegard M. Reznik-Schuller

Fig. 77. Poorly differentiated adenocarcinoma induced in a F344 rat by feeding diets containing p-cresidine for 2 years. Invasion into the bones of the ethmoid region is seen. Hand E, x 220

rna). This problem is compounded by the coexistence of epithelial and neurogenic cells in the ethmoid regions of the nasal cavities, as well as by the similarity in appearance of cytological elements and multicellular structures (such as rosettes) at the light microscopic level. Thus conclusive diagnosis without ultrastructural observations is very tenuous. Olfactory rosettes are usually round and are lined by well-differentiated tall columnar cells with basally located nuclei, which do not display marked cytologic atypia (Silva et al. 1983). The rosettes of poorly differentiated adenocarcinomas, on the other hand, are frequently composed of anaplastic cells exhibiting considerable pleomorphism (Reznik et al. 1980a, b). However, due to the absence of rosettes in some olfactory neuroblastomas (Elkon 1983), differential diagnosis at the light microscopic level should rely on the demonstration of neurofibrils by a suitable histochemical technique. Definitive differential diagnosis is facilitated by electron microscopic analysis. Ultrastructural features indicating a neurogenic origin include: in-

Fig.78. Invasion of an adenocarcinoma into the brain from the ethmoid region of a F344 rat fed diets containing p-cresidine for 2 years. Hand E, x 130

tercellular filamentous dendritic processes originating from tumor cell bodies, neurotubules, and intracellular dense-cored secretory granules (EIkon 1983). When these features are not demonstrated, a diagnosis of adenocarcinoma must be favored over one of neuroblastoma.

Biologic Features

The spontaneous occurrence of neoplasms in the nasal cavities of rats is extremely infrequent. In over 2500 male and female untreated F344 rats held for 2 years, only one benign and one malignant neoplasm were found in a single female rat, and these were in the respiratory (anterior) regions (Reznik et al. 1980a). No spontaneously occurring neoplasms have been reported in the ethmoid regions of the nasal cavities of rats. Treatment of rats with a wide variety of substances by various routes of exposure has been associated with the induction of neoplasms of the ethmoid regions. These have been comprehensively reviewed recently (Reznik-Schuller 1983 a; Stinson 1983). Several nitrosamines, given system-

Fig.79. Tumor induced in the olfactory region of a F344 rat by chronic inhalation of 1,2-dibromo-3-chloropropane. The cytoplasm is poorly differentiated, with ribosomes and polyribosomes predominating over other organelles. Electron micrograph, uranyl acetate and lead citrate, x 7000

ically, have been reported to induce neoplasms of the olfactory epithelium. Adenocarcinomas or olfactory neuroblastomas were diagnosed most frequently and squamous cell carcinomas were also reported. Some of the more potent compounds in this respect include N-nitroso-bis (2-hydroxypropyl)amine, N-nitrosomorpholine, N-nitroso-2, 6-dimethylmorpholine, N-nitrosopiperidine, Nnitrosomethylpiperazine, N-nitroso-3, 4-dichloropyrrolidine, and N-nitrosonornicotine. Other important environmental contaminants also inducing tumors of the ethmoid region include: dibromochloropropane and dibromoethane by inhalation and p-cresidine, procarbazine, 2-methoxy-5-methylanaline, and 1-methoxy-4-nitro-tetrachlorobenzene by systemic routes of exposure. Squamous cell carcinomas and adenocarcinomas or olfactory neuroblastomas are frequently induced by the same compound and may coexist in the same animal. In a comprehensive serial sacrifice experiment, the pathogenesis of tumors induced in the olfactory region of F344 rats by chronic treatment with


Fig.SO. Electron micrograph of tumor shown in Fig. 73. Luminal surfaces of tumor cells are lined by long slender microvilli, a marker of sustentacular cells. Uranyl acetate and lead citrate, x 10000

N-nitrosomethylpiperazine (0.13% in drinking water 5 days/week for life) was studied by light and electron microscopy (Reznik-SchUller 1983 b). Mter 4 weeks of treatment, focal hyperplasias (Fig. 81) developed in the basal layer of the olfactory epithelium. Ultrastructurally, some of these proliferated cells contained dense-cored granules of the neuroendocrine type (Fig. 82), while in others the fine structure of basal cells was evident. Around the 20th week of treatment, some of these lesions were found to have undergone endophytic growth, penetrating from the epithelium into the submucosa (Fig.83). From such lesions invasive carcinomas developed, which were composed of areas of variable morphological patterns. Electron microscopy revealed some cells with neuroendocrine features and others with elements of adenoid or squamous differentiation. In one case, axon-like cytoplasmic extensions indicated partial differentiation into neuroblasts. Similar changes were observed in rats following inhalation of dibromochloropropane or dibromoethane (Reznik et al. 1980 b).

52 Sherman F. Stinson and Hildegard M. Reznik-SchUller

Fig.81 (Above). Olfactory region in a F344 rat treated for 4 weeks with N-nitrosomethylpiperazine. Focal hyperplasia of small round to oval cells has developed in the basal epithelial layer. The surface of the lesion is covered by olfactory sensory and sustentacular cells. Toluidine blue, x 560

Fig. 82 (Below). Basal hyperplasia of cells of olfactory epithelium in a F344 rat treated for 4 weeks with N-nitrosomethylpiperazine. Some of the cells contain dense-cored cytoplasmic granules, the ultrastructural marker of neuroendocrine cells. Electron micrograph, uranyl acetate and lead citrate, x 10200

These observations are consistent with the conclusion that neoplasms in the ethmoid region are derived from multi potential basal cells in the olfactory epithelium, which are capable of various types of differentiation. Taken together with other information discussed previously, these data reinforce the opinion that a diagnosis of olfactory neuroblastoma as a tumor in this region should be made only with conclusive evidence of neurogenic origin.


Neoplasms of the ethmoid regions of the types described have been induced in hamsters, mice, and gerbils. Studies in various species using the same compound and similar dosing regimens suggest that the sensitivity of Syrian hamsters to nasal carcinogens is similar to that of rats, while European hamsters are more sensitive and Chinese hamsters and mice are less sensitive (ReznikSchuller 1983 a; Stinson 1983). Few studies have been conducted in other species, but some olfactory neoplasms have been reported. Adenocarcinomas in nonhuman primates and squamous cell carcinomas in dogs have been induced by nitrosamines (Reznik-Schuller 1983 a), and adenocarci-


nomas of the olfactory epithelium have been found in sheep (Young et al. 1961). Eleven nasal tumors classified as esthesioneuroepitheliomas were found among 21600 mammals necropsied at the Philadelphia Zoo (Montali et al. 1983). All were in small carnivores (raccoons, skunks, mink, etc.) and may have been related to housing on contaminated oak planks. As in rats, the spontaneous occurrence of nasal neoplasms in these species is extremely infrequent. In humans, tumors of the ethmoid regions are rare. Among these, squamous cell carcinomas predominate. Adenocarcinomas and olfactory neuroblastomas, with and without olfactory differentiation, have also been reported (Heffner 1983). The histological appearance of these tumors is similar to that found in rats.

Acknowledgement. This project has been funded in part with Federal funds from the Department of Health and Human Services, under contract number N01-CO-23909 with Litton Bionetics, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services; similarly, mention of trade names, commercial products, or organizations does not imply endorsement by the U. S. Government.

Fig.83. Olfactory region of a F344 rat treated for 20 weeks with N-nitrosomethylpiperazine. Proliferating cells from the basal layer of the olfactory epithelium have penetrated the submucosa. Toluidine blue, x 130

54 William D. Kerns

References

Elkon D (1983) Olfactory esthesioneuroblastoma. In: Reznik G, Stinson SF (eds) Nasal tumors in animals and man, vol II. Tumor pathology. CRC, Boca Raton, chap 6

Heffner DK (1983) Histopathologic classification of human sino nasal tumors. In: Reznik G, Stinson SF (eds) Nasal tumors in animals and man, vol II. Tumor pathology. CRC, Boca Raton, chap 1

Montali RJ, Valerio MG, Harshbarger JC (1983) Tumors of the nasal cavity in nondomesticated animals. In: Reznik G, Stinson SF (eds) Nasal tumors in animals and man, vol II. Tumor pathology. CRC, Boca Raton, chapt 11

Obert GJ, Devine KD, McDonald JR (1960) Olfactory neuroblastomas. Cancer 13: 205-215

Reznik G, Reznik-Schuller H, Ward JM, Stinson SF (1980a) Morphology of nasal-cavity tumours in rats after chronic inhalation of 1,2-dibromo-3-chloropropane. Br J Cancer 42: 772-781

Reznik G, Stinson SF, Ward JM (1980b) Respiratory pathology in rats and mice after inhalation of 1,2-dibromo-3-chloropropane or 1,2-dibromoethane for 13 weeks. Arch Toxicol46: 233-240

Reznik G, Reznik-Schiiller HM, Hayden DW, Russfield A, Murthy ASK (1981) Morphology of nasal cavity neoplasms in F344 rats after chronic feeding of p-cresidine, an intermediate of dyes and pigments. Anticancer Res 1 : 279-286

Reznik-Schiiller HM (1983 a) Nitrosamine-induced nasal cavity carcinogenesis. In: Reznik G, Stinson SF (eds)Nasal tumors in animals and man, vol III. Experimental nasal carcinogenesis. CRC, Boca Raton, chap 3

Reznik-Schuller HM (1983 b) Pathogenesis of tumors induced with N-nitrosomethylpiperazine in the olfactory region of the rat nasal cavity. JNCI 71: 165-172

Silva EG, Mackay B, Butler JJ (1983) Nasal neuroblastomas in man. In: Reznik G, Stinson SF (eds) Nasal tumors in animals and man, vol II. Tumor pathology. CRC, Boca Raton, chap 4

Stinson SF (1983) Nasal cavity cancer in laboratory animal bioassays of environmental compounds. In: Reznik G, Stinson SF (eds) Nasal tumors in animals and man, vol III. Experimental nasal carcinogenesis. CRC, Boca Raton, chap 7

Young S, Lovelace SA, Hawkins WW Jr, Catlin JE (1961) Neoplasms of the olfactory mucous membrane of sheep. Cornell Vet 51: 96-112

Squamous Cell Carcinoma, Nasal Mucosa, Rat

William D. Kerns

Synonym. Epidermoid carcinoma.

Gross Appearance

Clinically, squamous cell carcinomas, as well as all other malignant neoplasms of the nasal cavity, are invasive tumors that protrude dorsally or laterally from the nasal cavity (Fig.84). Frequently, the epidermis overlying the tumor is ulcerated and necrotic. Often, during the early stages of tumor growth, a unilateral nasoocular discharge is observed; this can be associated with tumor growth and interference with nasolacrimal drainage. Once the tumor has penetrated the nasal or maxillary bones, its growth is rapid. Moribund rats exhibit marked dyspnea and emaciation. Macroscopically, squamous cell carcinomas contain areas of whitish gray tissue and nests of laminated caseous material (keratin). The tumor is usually unilateral and may cause marked compression of the contralateral nasal passage (Fig. 85). Squamous cell carcinomas usually originate in the anterior nasal cavity (Kerns et al. 1983;

Lee and Trochimowicz 1982) and frequently extend posteriorly into the ethmoturbinates (Fig. 86) and olfactory bulbs.


Squamous cell carcinomas may originate from metaplastic squamous epithelium or from the squamous epithelium that is normally found in the nasal vestibule. The nasoturbinates, maxilloturbinates, nasal septum, or lateral wall may be affected. While invasion of the nasal and maxillary bones or nasal septum is common, tumor growth through the hard palate into the oral cavity is rare. Tumor cell differentiation varies widely and many nasal squamous cell carcinomas are osteolytic. With this type of nasal cancer, as with many others, poor differentiation is associated with invasion of the nasal bone (Lee and Trochimowicz 1982). However, some of the most differentiated carcinomas may also be very invasive (Fig. 87). Excessive keratinization (Fig. 88) and extension of tumor tissue into the ethmoturbinates and naso-

pharyngeal duct are common histomorphological findings, but extension into the brain (Fig. 89) is rare (less than 2%) (Pavkov et al. 1981; Lee and Trochimowicz 1982). When metastases do occur, they are found in the regional lymph nodes and lung. Cytologically, well-differentiated carcinomas are characterized by prominent intracellular bridges, normal keratinization (although abundant in most cases), minimal nuclear atypia, a low mitotic index, and invasion of adjacent structures. Some squamous cell carcinomas are characterized by foci of less differentiated tumor cells (Fig. 90). Although keratin (intracellular and extracellular) is normally present in these foci, the tumor cells do not resemble mature squamous cells. They are recognized by their nuclear atypia, abnormal keratinization (parakeratosis), polygonal and epithelioid morphology, abnormal mitotic figures, and absence of intracellular bridges.

Ultrastructure

The cells of well-differentiated squamous carcinomas are recognized by the presence of tonofilaments, desmosomes, prominent nucleoli, and tubular profiles of rough endoplasmic reticulum (Figs.91 and 92). Cytoplasmic membranes are characterized by elaborate intercellular interdigitations. Less differentiated tumors demonstrate a marked decrease in the number of glycogen particulates, desmosomes, tonofilaments, and cell membrane interdigitations (Lee et al. 1983).


Squamous cell carcinomas should not pose any diagnostic problems. Some less differentiated carcinomas, especially those with spindle-shaped cells and acinar structures, may require ultrastructural, immunoultrastructural, or immunohistochemical (keratin) verification as to the cell of origin (Nagle et al. 1983; N athrath et al. 1982; Wilson et al. 1982).

Biologic Features

With one exception (Lee et al. 1983), squamous cell carcinomas are known to occur only as experimentally induced lesions of the nasal cavity in the rat. They have been induced with a variety of related and unrelated compounds by inhalation, as well as by oral, intraperitoneal, and subcutane-

Squamous Cell Carcinoma, Nasal Mucosa, Rat 55

Fig.84. A rat exposed to formaldehyde. The large subcutaneous mass anterior to the eyes is an invasive squamous cell carcinoma of the nasal mucosa

Fig.85. Coronal section of an undeca1cified head from a rat exposed to formaldehyde. An invasive squamous cell carcinoma protrudes from the nasal cavity and has compressed the contralateral nasal passage

56 William D. Kerns

Fig.86. Midsagittal section of a rat exposed to formaldehyde. A destructive squamous cell carcinoma of the anterior nasal cavity extends into the ethmoturbinates (arrow)

Fig. 87. Advanced squamous cell carcinoma that has invaded the nasal bone of a rat exposed to formaldehyde. Hand E, x 180 (reduced by 15%)

ous routes of exposure (Table 3; Stinson 1983). The pathogenesis of this lesion has not been totally defined, but necrosis and/or hyperplasia of the respiratory epithelium followed by epidermoid (squamous) metaplasia and dysplasia usually precede neoplasia (Pavkov et al. 1981; Kerns et al. 1983).


In humans, squamous cell cell carcinoma is the predominant malignant tumor of the anterior na-

sal cavity (Ash et al. 1964). Nuclear pleomorphism is uncommon and the tumors may show slight or usually no evidence of keratinization. Squamous cell carcinomas in this location rarely metastasize. Workers in the furniture and leather industry are reported to have a higher incidence of nasal cancer (Roush et al. 1980; Andersen et al. 1977; Brinton et al. 1977; Collan 1983; Buiatti et al. 1983), and a nasal squamous cell carcinoma has been reported in a patient with a history of formaldehyde exposure (Halperin et al. 1983).

Fig.88. A squamous cell carcinoma, characterized by excessive keratinization, has replaced the nasoturbinate in this rat exposed to formaldehyde. H and E, x 90 (reduced by 15%)

Fig.89. An invasive squamous cell carcinoma has totally replaced the left olfactory bulb in this rat exposed to formaldehyde. Hand E, x 90 (reduced by 15%)


58 William D.Kems

Fig.90 (Above). A poorly differentiated squamous cell carcinoma of the nasal cavity in a rat exposed to formaldehyde. Hand E, x 360

Fig.91 (Below). Neoplastic squamous cells from a nasal carcinoma. TEM, x 6000


Table 3. Squamous cell carcinomas observed in the nasal cavity of the rat

Chemical Strain Malea Femalea Study type Reference

Formaldehyde F344 Inhalation Kerns et al. 5.6 ppm 1/119 1/116 1983

14.3 ppm 51/117 52/115

Phenylglycidyl ether S-D Inhalation Lee et al. o ppm 1/89 0/87 1983

12 ppm 9/85 4/89

p-Cresidine F344 2/54 8/47 Feeding Reznik et al. 1.0% 1981

1,2-Dibromo-3-chloropropane F344 Inhalation Reznik et al. 0.6 ppm 4/50 5/50 1980a,b 3.0 ppm 17/45 5/49

Epichlorohydrin S-D Inhalation Laskin et al. 30 ppm 1/100 ND 1980

100 ppm 15/140 ND

Formaldehyde and hydrogen S-D Inhalation Albert et al. chloride 1982

14.7 ppm HCHO and 25/99 ND 10.6 ppm HCl

Hexamethylphosphoramide S-D Inhalation Lee and Trochimowicz 50ppb 24/194b 1982

100ppb 59/200b

400ppb 1371219b

4000ppb 1201215b

Dioxane S-D Drinking Hoch-Ligeti 0.75% 1/30 ND water et al. 1970 1.00% 1/30 ND 1.40% 2/30 ND 1.80% 2/30 ND

Bis( chloromethyl)ether S-D Inhalation Kuschner 0.1 ppm 1/50 ND et al. 1975

l,4-Dioxane Sherman Drinking Kociba et al. 1.0% 1/60 2/60 water 1974

Dimethylcarbamoylchloride S-D Inhalation Sellakumar 1 ppm 96% ND et al. 1980

Phenacetin S-D Feeding Isaka et al. 1.25% 3/22 1/25 1979 2.50% 2127 0127

3,4,5-Trimethoxycinnamaldehyde NR Injection Schoental and Gibbard 150 mg/kg (IP) and 2/4 ND 1972 100 mg/kg (SC)

S-D, Sprague-Dawley; ND, not determined; NR, not reported a No. of tumors/no. of nasal cavities evaluated b Sexes were combined

60 William D.Kerns

In other laboratory animals, chemically induced squamous cell carcinomas have been reported in the mouse (Kerns et al. 1983; Reznik et al. 1980 b), hamster (Sellakumar et al. 1980; Feron et al. 1982), and gerbil (Cardesa et al. 1976). Squamous cell carcinomas have been reported in other species but are not known to be associated with chemical or infectious agents. In the dog, the males of dolichocephalic breeds are most often affected, and squamous cell carcinomas were reported to account for 33% of all nasal and paranasal-sinus carcinomas in one study (Moulton 1978) and 8% of all nasal and paranasal-sinus neoplasms in another study (Madewell et al. 1976). In the cat, squamous cell carcinoma was the most frequently reported nasal neoplasm, with reported frequencies of 71 % (Moulton 1978) and 44% (Madewell et al. 1976). Squamous cell carcinoma originating in the nasal cavity and sinuses of the horse has also been reported (Jean and Daudel 1949; Cotchin 1967), and was found to be the most common nasal neoplasm (five of 13) in one study (Madewell et al. 1976).

Fig.92. Neoplastic squamous cells from a nasal carcinoma. Note desmosomes (D) and bundles of to no filaments (T). TEM, x 24000 (reduced by 15%)

References

Albert RE, Sellakumar AR, Laskin S, Kuschner M, Nelson N, Snyder CA (1982) Gaseous formaldehyde and hydrogen chloride induction of nasal cancer in rats. JNCI 68: 597-603

Andersen HC, Andersen I, Solgaard J (1977) Nasal cancers, symptoms and upper airway function in woodworkers. Br J Ind Med 34: 201-207

Ash JE, Beck MR, Wilkes JD (1964) Atlas of tumor pathology. Tumors of the upper respiratory tract and ear. Armed Forces Institute of Pathology, Washington, DC, sect IV, fasc 12 and 13

Brinton LA, Blot WJ, Stone BJ, Fraumeni JF Jr (1977) A death certificate analysis of nasal cancer among furniture workers in North Carolina. Cancer Res 37: 3473-3474

Buiatti E, Geddes M, Carnevale F, Merler E (1983) Nasal cavity and paranasal sinus tumors in woodworkers and shoemakers in Italy compared to other countries. In: Reznik G, Stinson SF (eds) Nasal tumors in animals and man, vol 1. Anatomy, physiology and epidemiology. CRC, Boca Raton, chap 5

Cardesa A, Pour P, Haas H, Althoff J, Mohr U (1976) Histogenesis of tumors from the nasal cavities induced by diethylnitrosamine. Cancer 37: 346-355

Collan Y (1983) Epidemiologic and etiologic aspects and histopathology of nasal carcinoma in Finland. In:

Reznik G, Stinson SF (eds) Nasal tumors in animals and man, vol 1. Anatomy, physiology and epidemiology. CRC, Boca Raton, chap 4

Cotchin E (1967) Spontaneous neoplasms of the upper respiratory tract in animals. In: Muir CS, Shanmugaratnam K (eds) Cancer of the naso-pharynx. International Union Against Cancer, Monogr series no 1, Medical Examination, Flushing, pp 203-215

Feron VJ, Kruysse A, Wouters en RA (1982) Respiratory tract tumours in hamsters exposed to acetaldehyde vapour alone or simultaneously to benzo( a )pyrene or diethylnitrosamine. Eur 1 Cancer Clin Oncol18: 13-31

Halperin WE, Goodman M, Stayner L, Elliott LJ, Keenlyside RA, Landrigan Pl (1983) Nasal cancer in a worker exposed to formaldehyde. lAMA 249: 510-512

Hoch-Ligeti C, Argus MF, Arcos lC (1970) Induction of carcinomas in the nasal cavity of rats by dioxane. Br 1 Cancer 24: 164-167

Isaka H, Yoshii H, Otsuji A, Koike M, Nagai Y, Koura M, Sugiyasu K Kanabayashi T (1979) Tumors of SpragueDawley rats induced by long-term feeding of phenacetin. Gan 70: 29-36

Jean R, Daudel R (1949) Epithelioma des sinus et des fosses nasales chez un cheval. Bull Serv Elev Indust Anim AOF 2: 15-21

Kerns WD, Pavkov KL, Donofrio DJ, Gralla EJ, Swenberg lA (1983) Carcinogenicity of formaldehyde in rats and mice after long-term inhalation exposure. Cancer Res 43: 4382-4392

Kociba RJ, McCollister SB, Park C, Torkelson TR, Gehring Pl (1974) 1,4-Dioxane. I. Results of a 2-year ingestion study in rats. Toxicol Appl Pharmacol 30: 275-286

Kuschner M, Laskin S, Drew RT, Cappiello V, Nelson N (1975) Inhalation carcinogenicity of alpha halo ethers. III. Lifetime and limited period inhalation studies with bis( chloromethyl)ether at 0.1 ppm. Arch Environ Health 30:73-77

Laskin S, Sellakumar AR, Kuschner M, Nelson N, La Mendola S, Rusch GM, Katz GV, Dulak NC, Albert RE (1980) Inhalation carcinogenicity of epichlorohydrin in noninbred Sprague-Dawley rats. JNCI 65: 751-757

Lee KP, Trochimowicz HJ (1982) Induction of nasal tumors in rats exposed to hexamethylphosphoramide by inhalation. JNCI 68: 157-171

Lee KP, Schneider PW, Trochimowicz HJ (1983) Morphologic expression of glandular differentiation in the epidermoid nasal carcinomas induced by phenylglycidyl ether inhalation. Am J Pathol 111: 140-148

Madewell BR, Priester W A, Gillette EL, Snyder SP (1976) Neoplasms of the nasal passages and paranasal sinuses in domesticated animals as reported by 13 veterinary colleges. Am J Vet Res 37: 851-856


Moulton JE (1978) Tumors of the respiratory system. In: Moulton JE (ed) Tumors in domestic animals. University of California Press, Berkeley, chap 6

Nagle RB, McDaniel KM, Clark VA, Payne CM (1983) The use of antikeratin antibodies in the diagnosis of human neoplasms. Am J Clin Pathol 79: 458-466

Nathrath WB, Wilson PD, Trejdosiewicz LK (1982) Immunohistochemical localisation of keratin and luminal epithelial antigen in myoepithelial and luminal epithelial cells of human mammary and salivary gland tumours. Pathol Res Pract 175: 279-288

Pavkov KL, Kerns WD, Mitchell RI, Connell MM, Donofrio DJ, Harroff HH (1981) A chronic inhalation toxicology study in rats and mice exposed to formaldehyde. In: Chemical Industry Institute of Toxicology Final Report, Dockett no. 10922 Battelle, Columbus Labs, Columbus

Reznik, G, Reznik-Schuller HM, Hayden DW, Russfield A, Murthy ASK (1981) Morphology of nasal cavity neoplasms in F344 rats after chronic feeding of p-cresidine, an intermediate of dyes and pigments. Anticancer Res 1 : 279-286

Reznik G, Reznik-Schuller HM, Ward 1M, Stinson SF (1980a) Morphology of nasal-cavity tumours in rats after chronic inhalation of 1,2-dibromo-3-chloropropane. Br J Cancer 42: 772-781

Reznik G, Ulland B, Stinson SF, Ward 1M (1980b) Morphology and sex-dependent manifestation of nasal tumors in B6C3Fl mice after chronic inhalation of 1,2-dibromo-3-chloropropane. 1 Cancer Res Clin Oncol 98:75-83

Roush GC, Meigs lW, Kelly JA, Flannery JT, Burdo H (1980) Sinonasal cancer and occupation: a case-control study. Am 1 Epidemiol111: 183-193

Schoental R, Gibbard S (1972) Nasal and other tumours in rats given 3,4,5,-trimethoxy-cinnamaldehyde, a derivative of sinapaldehyde and other alpha, beta-unsaturated aldehydic wood lignin constituents. Br J Cancer 26: 504-505

Sellakumar AR, Laskin S, Kuschner M, Rusch G, Katz GV, Snyder CA, Albert RE (1980) Inhalation carcinogenesis by dimethy1carbamoyl chloride in Syrian golden hamsters. J Environ Pathol Toxicol4: 107-115

Stinson SF (1983) Nasal cavity cancer in laboratory animal bioassays of environmental compounds. In: Reznik G, Stinson SF (eds) Nasal tumors in animals and man, vol III. Experimental carcinogenesis, CRC, Boca Raton, chap 7

Wilson PD, Nathrath WB, Trejdosiewicz LK (1982) Immunoelectron microscopic localisation of keratin and luminal epithelial antigens in normal and neoplastic urothelium. Pathol Res Pract 175: 289-298

62 Parviz M. Pour

Squamous Cell Carcinoma, Upper Respiratory Tract, Syrian Hamster

Parviz M. Pour

Synonyms. Epidermoid carcinoma; squamous cell tumor.

Gross Appearance

In its earliest stages, a squamous cell carcinoma is not visible grossly. Larger lesions may elevate the mucosa or project from its surface but are not distinctive in any gross features. Invasive tumors appear as grayish white masses penetrating into the surrounding tissue.


This neoplasm arises from the epithelium or from the columnar epithelium of ducts or glands just beneath the surface epithelium in the mucosa of the nasal cavity. The initial change in the respiratory epithelium is disorganization of the epithelial cells followed by a gradual squamous metaplasia. Increasing focal or multifocal atypia of the epithelium may reach an extent consistent with carcinoma in situ. The metaplastic epithelium sometimes continues to grow and project from the surface to form papillomas. Early squamous cell carcinoma is recognized by the accumulation of many layers of atypical squamous epithelial cells which have lost polarity and replaced the respiratory epithelial cells. These squamous cells initially respect the basal laminae, but eventually penetrate them (Figs.93 and 94) and sometimes invade underlying mucous glands. Invasion is evident by small epithelial nests (Fig. 95), which appear as isolated islands beneath the surface epithelium and invade lymphatics or veins (Fig.96). The origin of these malignant epithelial nests cannot always be determined; they may come from the surface epithelium or the submucosal glands.

Ultrastructure

The ultrastructural features of this neoplasm are similar to those of squamous cell carcinomas of the lung in Syrian hamsters (discussed on page 116).


Most squamous cell carcinomas are well-differentiated keratinizing types. However, various degrees of differentiation are encountered (Figs. 97 and 98). In tumors with a mixture of glandular and squamous epithelium, a diagnosis of mucoepidermoid tumor is justified if both squamous and glandular cells (secreting PAS-positive material) are present in adequate proportions and in a mixed pattern. Squamous cell carcinoma in the upper respiratory tract must be differentiated from squamous papilloma and squamous metaplasia (described in detail on page 36). Squamous papillomas project from the surface of the respiratory epithelium, resemble normal stratified squamous epithelium, and do not undergo the loss of polarity, dysplasia, and growth through the basal lamina which are characteristic of squamous cell carcinomas. Hyperplasia may be seen in respiratory epithelium with the characteristic increase in number of cells and may accompany squamous metaplasia, in which the pseudostratified columnar cells of the respiratory mucosa are replaced by squamous cells. Papillary projections into the lumen and downgrowth through the basal lamina do not occur. Squamous metaplasia may occur in association with chronic inflammation and does not necessarily precede neoplasia.

Biologic Features

Natural History. Squamous cell metaplasia of the nasal respiratory epithelium, especially in the anterior nasal region, is common in several hamster strains (Pour et al. 1976b, 1979). Spontaneous squamous cell tumors are extremely rare. Thus far, in a large number of cases studied, we have seen no carcinomas and only one papilloma in the maxillary turbinate of an albino hamster (Pour 1983), a finding that could indicate either that metaplasia in hamsters does not present a premalignant lesion or that the time sequence between metaplasia and malignancy is longer than the usual hamster life span. We tend to believe the first to be true, since metaplasia occurs often during the inflammatory process.

Squamous Cell Carcinoma, Upper Respiratory Tract, Syrian Hamster 63

Fig. 93 (Above). Early squamous cell carcinoma induced by BHP. Early invasion of the basal membrane (arrows). H and E, x 195 (reduced by 30%)

Most squamous cell carcinomas and mucoepidermoid tumors are invasive initially, rather than expansive (Fig.98). At certain points, the exophytic (expansive) growth begins to invade rapidly, primarily in the bones, and these tumors occasionally present bulky, fungating masses which extend to the external nares. For some as yet unknown reason, they metastasize infrequently, despite their considerable size. Only rarely do tumors show early invasion of lymphatics and blood vessels (Fig.96) and metastasize to the submandibular lymph nodes.

Fig.94 (Below). Dysplastic squamous cell epithelium (bottom) and early squamous cell carcinoma invading the submucosa (arrows) in a MPN-treated hamster. Hand E, x 195 (reduced by 30%)

Etiology. Certain nitrosamines are potent inducers of paranasal cavity tumors. Remarkably, the morphology of induced lesions can vary from one compound to another. Some of these compounds (Table 1 p.28), such as N-nitrosomethyl(2-oxobutyl)amine (M-2-0B) (Pour et al. 1983) and N-nitrosohexamethyleneimine (N-6-MI) (Althoff et al. 1973), induce adenocarcinomas with a few squamous cell tumors. In contrast, N-nitrosobis(2-hydroxypropyl)amine (BHP) (Pour et al. 1975), Nnitrosodi-n-propylamine (DPN) (Pour et al. 1973, 1974b), N-nitrosomethyl-n-propylamine (MPN)

64 Parviz M. Pour

Fig.95 (Above). Malignant squamous cells (arrows) in the submucosa of the anterior nasal cavity adjacent to hyperplastic and metaplastic respiratory epithelium. Hand E, x 195 (reduced by 30%)

(Pour et al. 1974 d), 1-acetoxypropylnitrosamine (l-APPN) (Althoff et al. 1977 a), N-nitrosovinylethylamine (VEN) (Althoff et al. 1977b), N-nitrosomethyl(2-oxopropyl)amine (MOP) (Pour et al. 1980), and N-nitrosobis(2-acetoxypropyl)amine (BAP) (Pour et al. 1976a) are potent inducers of squamous cell neoplasms.

Pathogenesis. Squamous cell carcinoma induction was found in most instances to follow squamous cell metaplasia, hyperplasia, dysplasia and in situ

Fig. 96 (Below). Squamous cell carcinoma induced by DPN in the anterior nasal cavity with invasion of lymphatic (straight arrow) and blood vessels (curved arrows). Hand E, x 390 (reduced by 30%)

changes. However, this neoplasm evidently can also develop de novo after high doses of potent carcinogens. The gradual changes in respiratory epithelium can best be demonstrated by serial sacrifice of animals during the study. The earliest alterations are characterized by focal proliferation of epithelial cells, which may undergo atypia followed by squamous cell metaplasia. Whereas metaplastic epithelium sometimes continues to grow and to form small or bulky papillomas, in others increasing focal or multifocal atypia of the

Squamous Cell Carcinoma, Upper Respiratory Tract, Syrian Hamster 65

Fig. 97 (Above). Invasive squamous cell carcinoma induced by 2-0PPN in anterior nasal cavity. No basal membrane could be found around this cellular mass by special stains. Hand E, x 195

Fig.98 (Below). Fairly well-differentiated fungating squamous cell carcinoma induced by BHP. Tumor originates from the maxillary turbinate mucosa and invades the submucosa (arrows). Hand E, x 72

66 Parviz M. Pour

epithelium to degrees consistent with carcinoma in situ ensue. At this stage the originally flat epithelium becomes exophytic and/or endophytic and (in the latter case) with more or less broad papillae that initially respect the basal laminae. Atypical epithelium often grows into or develops within the submucosal glands and appears invasive.

Location. Squamous cell tumors are found primarily in the anterior regions, mostly on the nasoturbinates and maxilloturbinates. Squamous cell carcinomas have also occurred in much lower incidences in the posterior nasal cavity (Pour et al. 197 4 b), primarily at the floor or ceiling level of the ethmoid region. We have found no tumors that originate in the middle nasal cavity.

Frequency. Although no sexual differences were observed in many of our studies, a male predominance was seen in a study using N-6-MI, with a male: female ratio of 2.5: 1 (Althoff et al. 1973). The male preponderance appears to have a significant biologic value, since it correlates with the situation in man and in rats, in which castration inhibited nasal cancer induction by one of the potent nasal carcinogens (Pour and G6tz 1983). The possible sex hormone dependence of nasal tumors is yet to be determined. The incidence of each histologic tumor type appears to depend on the nature of the carcinogen. A high incidence of squamous cell carcinomas has been seen after BHP (Pour et al. 1975) and MPN treatment (Pour et al. 1974d), whereas DPN (Pour et al. 1973), N-nitroso-2-hydroxypropyl-npropyl amine (2-HPPN) (Pour et al. 1974c) and N-nitroso-2-oxopropyl-n-propylamine (2-0PPN) (Pour et al. 1974a) primarily induce mucoepidermoid neoplasms in as many as 71 % of the animals. The incidence and mUltiplicity of squamous cell and mucoepidermoid tumors depend on the nature of the carcinogen, its dose, duration of treatment, and the method employed for histological examination to allow proper recognition of the lesions (Pour et al. 1976c).

References

Althoff J, Cardesa A, Pour P, Mohr U (1973) Carcinogenic effect of n-nitrosohexamethyleneimine in Syrian golden hamsters. JNCI 50: 323-329

Althoff J, Grandjean C, Pour P, Gold B (1977 a) Local and systemic effects of 1-acetoxypropylnitrosamine in Syrian golden hamsters. Z Krebsforsch 90: 127-140

Althoff J, Grandjean C, Russell L, Pour P (1977b) Vinylethylnitrosamine: a potent respiratory carcinogen in Syrian hamsters. JNCI 58: 439-442

Pour PM (1983) Spontaneous respiratory tract tumors in Syrian hamsters. In: Reznik-Schuller HM (ed) Comparative respiratory tract carcinogenesis, vol I. Spontaneous respiratory tract carcinogenesis. CRC, Boca Raton, chap '7

Pour PM, Gotz U (1983) Prevention of N-nitrosobis(2-oxopropyl)amine-induced nasal cavity tumors in rats by orchiectomy. JNCI 70: 353-357

Pour P, KrUger FW, Cardesa A, Althoff J. Mohr U (1973) Carcinogenic effect of di-n-propylnitrosamine in Syrian golden hamsters. JNCI 51: 1019-1027

Pour P, Althoff J, Cardesa A, KrUger F, Mohr U (1974a) Effect of beta-oxidized nitrosamines on Syrian golden hamsters. II. 2-0xopropyl-n-propylnitrosamine. JNCI 52: 1869-1874

Pour P, Cardesa A, Althoff 1. Mohr U (1974b) Tumorigenesis in the nasal olfactory region of Syrian golden hamsters as a result of di-n-propylnitrosamine and related compunds. Cancer Res 34: 16-26

Pour P, KrUger FW, Althoff J, Cardesa A, Mohr U (1974c) Effect of beta-oxidized nitrosamines on Syrian golden hamsters. I. 2-Hydroxypropyl-n-propylnitrosamine. JNCI 52: 1245-1249

Pour P, Kruger FW, Cardesa A. Althoff J, Mohr U (1974d) Tumorigenicity of methyl-n-propylnitrosamine in Syrian golden hamsters. JNCI 52: 457-462

Pour P, KrUger FW, Althoff J, Cardesa A, Mohr U (1975) Effect of beta-oxidized nitrosamines on Syrian hamsters. III. 2,2'-Dihydroxy-di-n-propylnitrosamine. JNCI 54:141-146

Pour P, Althoff J, Gingell R, Kupper R, KrUger F, Mohr U (1976a) N-nitroso-bis(2-acetoxypropyl)amine as a further pancreatic carcinogen in Syrian golden hamsters. Cancer Res 36: 2877-2884

Pour P, Mohr U, Cardesa A, Althoff J, Kmoch N (1976b) Spontaneous tumors and common diseases in two colonies of Syrian hamsters. II. Respiratory tract and digestive system. JNCI 56: 937-948

Pour P, Stanton MF, Kuschner M, Laskin S, Shabad LM (1976 c) Tumours of the respiratory tract. In: Turusov VS (ed) Pathology of tumours in laboratory animals, vol 1. Tumours of the rat, part 2. IARC Sci Publ 6: 1-40

Pour P, Althoff J, Salmasi SZ, Stepan K (1979) Spontaneous tumors and common diseases in three types of hamsters. JNCI 63: 797-811

Pour P, Gingell R, Langenbach R, Nagel D, Grandjean C, Lawson T, Salmasi S (1980) Carcinogenicity of N-nitrosomethyl(2-oxopropyl)amine in Syrian hamsters. Cancer Res 40: 3585-3590

Pour PM, Nagel D, Lawson T (1983) Carcinogenicity of Nnitrosomethyl(2-oxobutyl)amine and N-nitrosomethyl(3-oxobutyl)amine in Syrian hamsters with special reference to the pancreas. Cancer Res 43: 4885-4890

Adenocarcinoma, Anterior Nasal Epithelium, Rat 67

Adenocarcinoma, Anterior Nasal Epithelium, Rat

Sherman F. Stinson and Gerd Reznik

Gross Appearance

In the rat, adenocarcinomas of the upper respiratory epithelium are found almost exclusively in the nasal cavities, few having been reported in the larynx or trachea. Adenocarcinomas in the anterior nasal cavities arise most frequently from the epithelium lining the naso- and maxilloturbinals and the nasal septum (Fig. 99). Derivation from the epithelium lining the lateral walls is less common. The neoplasms appear as pink to gray sessile, exophytic masses protruding from the turbinals or septum and varying in size from less than a millimeter in diameter to large masses which en-

Fig.99. Cross section through anterior nasal cavities of a F344 rat following inhalation of dibromochloropropane for 106weeks. A large adenocarcinoma originating from the maxilloturbinal (a) and multiple small adenomas of the nasoturbinals (b) and dorsal wall (c) are seen. Hand E, x 5.5

tirely fill the nasal passage, compress the turbinals, and displace the nasal septum. Occasionally, adenocarcinomas invade through the bony encasement of the nose and become grossly visible on the external dorsolateral surface as a bulging subcutaneous mass (Fig. 100). As these neoplasms are almost always the result of exposure to nasal carcinogens, their spontaneous occurrence being extremely rare, multiple tumors are not an uncommon finding, and they may also coexist with other nasal tumors, including adenomas, papillomas, and squamous cell carcinomas.

Fig. tOO. Cross section through anterior nasal cavities of a F344 rat. Dibromochloropropane inhaled for 106 weeks. A large adenocarcinoma has invaded through the dorsal and lateral walls. Hand E, x 5

68 Sherman F. Stinson and Gerd Reznik


A broad and continuous range of degrees of differentiation, from well-differentiated adenomas and low-grade adenocarcinomas to relatively poorly differentiated highly malignant adenocarcinomas, can arise from the nasal respiratory epithelium. Glandular tumors of the nasal turbinates are usually adenomas and well-differentiated adenocarcinomas, while more poorly differentiated adenocarcinomas are found most frequently in the epithelium over the maxilloturbinals, dorsal nasal meatus, and concha. Well-differentiated tumors are formed of cords or sheets of uniform eosinophilic cells with round to oval nuclei, arranged in well-formed glandular and cystic structures (Figs. 101 and 102). These glandular structures are commonly filled with an amorphous, PAS-positive material or cellular debris. The nuclei have peripherally localized chromatin and, usually, a centrally located nucleus. Mitotic figures are not frequent and are usually normal in

Fig. tOt. Moderately well-differentiated adenocarcinoma (a) of the mucosa of the nasoturbinal in a F 344 rat fed pcresidine for 2 years. Hand E, x 130

type when found. Invasion is usually minimal in well-differentiated tumors and consists of cords or nests of cells penetrating into the submucosa adjacent to the nasal turbinal or nasal septum and occasionally eroding these structures. The poorly differentiated adenocarcinomas have a more solid appearance and are composed of cells with some cellular and nuclear pleomorphism (Figs. 103 and 104). Completely to incompletely formed acinar structures are surrounded by disorganized sheets and cords of round to elongated cells with widely varying nuclear cytoplasmic ratios (Fig. 104). A relatively high rate of cell division is evidenced by numerous mitotic figures. Erosion of the bony nasal structures and invasion of the tissues of the nasal well are common and often extensive with large neoplasms (Fig. 100). While most of the nasal adenocarcinomas have a predominant degree of differentiation, areas of well or poorly differentiated architecture can usually be found within a single tumor, making

Fig.t02. Higher magnification of an area from the adenocarcinoma pictured in Fig. lOt. Hand E, x 330

classification of the degree of differentiation of the neoplasm difficult (Lee et al. 1983).

Ultrastructure

Adenocarcinomas of the nasal respiratory epithelium have not been studied by means of electron microscopy in the rat or other laboratory rodents.


It is difficult to distinguish a well-differentiated adenocarcinoma from an adenoma of the nasal epithelium. Their localization and cell of origin are identical, and the size of the tumor, pattern of growth, and histologic appearance are often very similar. Criteria for malignancy include demonstration of invasion into the submucosa, bones, or

Fig.103. Papilloma (a) of the nasoturbinal and adenocarcinoma (b) of the maxilloturbinal in a F 344 rat that inhaled dibromochloropropane for 2 years. Hand E, x 35


cartilage and the presence of traditional cytologic changes, including anaplasia, pleomorphism, and abundant or abnormal mitoses. Another diagnostic problem arises in distinguishing between poorly differentiated adenocarcinomas of the respiratory epithelium and adenocarcinomas of the olfactory epithelium (see page 47). Both are similar in histologic appearance and can be induced by the same compounds. The close spatial relationship of the ethmoid and nasal regions, combined with the infiltrative and expansive behavior of the neoplasms, can create confusion as to the site of origin. Multiple or serial sectioning will often give enough information to resolve the problem. Adenocarcinomas of the olfactory epithelium are usually more anaplastic than those of the respiratory epithelium, being composed of cells with scant cytoplasm and very pleomorphic nuclei with large nucleoli. PAS-positive material has not been reported in the aci-

Fig.104. Higher magnification of an area from the adenocarcinoma in Fig.t03. Note relatively solid pattern of poorly differentiated cells, complete and incomplete formation of glands, and a large number of mitotic figures. H and E. x 220

70 Sherman F. Stinson and Gerd Reznik

nar structures of tumors of the olfactory epithelium. Ultrastructurally, adenocarcinomas of the olfactory epithelium reportedly contain cells showing features of sustentacular cells, such as smooth endoplasmic reticulum (see page 8). These would not be expected in cells of adenocarcinomas of the respiratory epithelium, although they are yet to be studied with the electron microscope. Finally, olfactory adenocarcinomas usually penetrate through the cribriform plate into the brain, while those arising from the respiratory epithelium invade laterally through the nasal and maxillary bones. Other tumors of these regions (squamous cell carcinoma, papilloma, mucoepidermoid tumor, hemangioma, and hemangiosarcoma) are easily distinguishable on the basis of histologic characteristics.

Fig.105. Nasoturbinal of a F344 rat fed p-cresidine for 75 weeks. Note goblet cell hyperplasia and squamous metaplasia of the epithelium and submucosal glands. H and E, x 130

Biologic Features

The spontaneous occurrence of adenocarcinomas of the upper respiratory epithelium in rats is very low. In a series of 1794 male and 1754 female untreated control F344 rats held for 2 years, only one adenocarcinoma of the trachea was found, and none were found in the larynx or nasal cavities (Goodman et al. 1979). Gross examinations were completed in this study but histologic sections through the nose were not made. In a later report, one adenocarcinoma in the anterior nasal cavity of an untreated control female F344 rat has been described (Reznik et al. 1980a). Several compounds have been associated with the induction of adenocarcinomas of the nasal respiratory epithelium; these were comprehensively reviewed recently (Reznik et al. 1981; ReznikSchuller 1983; Stinson 1983). These compounds include several different nitrosamines and other environmental substances. Nitrosamines have

Fig.106. Nasoturbinal of a F344 rat fed p-cresidine for 75 weeks. Note squamous metaplasia of the epithelium (a) and dysplasia and squamous metaplasia of the submucosal glands (b). Hand E, x 330

generally been administered systemically, while other substances have induced nasal adenocarcinomas by inhalation (dibromochloropropane, dibromo ethane) or when given in the feed (p-cresidine, 2-methoxy-5-methylanaline, phenacetin). Most of these compounds also induce benign nasal neoplasms (papillomas, papillary adenomas) and other malignant nasal neoplasms (squamous cell carcinoma, poorly differentiated adenocarcinoma of the ethmoid epithelium). Any of these neoplasms may coexist in the same animal after exposure to these carcinogens. Early morphological events associated with carcinogenesis in the nasal respiratory epithelium include a reaction of the epithelium typified by disorientation of basal and ciliated cells, loss of cilia, and cytomegaly of basal cells, followed by focal hyperplasia, mucous or glandular metaplasia, and squamous metaplasia (Figs. 105 and 106) (Reznik et al. 1980 b). Many of these changes are also observed in the submucosal glands of the anterior nasal cavities. The earliest neoplasms observed are papillomas and papillary adenomas. The similarity in distribution of the adenomas and adenocarcinomas, the continuum of histologic features from adenomas to poorly differentiated adenocarcinomas, and the fact that they are induced by the same compounds make it tempting to speculate that the adenomas have a malignant potential, although no direct observations have been reported to support this hypothesis.


Adenocarcinomas of the nasal respiratory epithelium have been induced in mice and hamsters (Reznik-Schuller 1983; Stinson 1983). These neoplasms are similar in histologic appearance and distribution to those found in the rat and are induced by the same compounds. Syrian hamsters appear to have a sensitivity similar to rats to nasal respiratory carcinogenesis, while European hamsters are more sensitive and Chinese hamsters and mice less sensitive. These differences, in some cases, may be due to differences in main airstream flow and mucociliary clearance (Schreider 1983). Well-differentiated adenocarcinomas similar to those found in rats have been reported in sheep (Njoku and Chineme 1983). These neoplasms may have an epidemiologic association with aflatoxin-contaminated feed. Nasal respiratory adenocarcinomas also occur in dogs (Patniak 1983) with similar geographic and temporal epidemio-


logic patterns to those observed in man (Hayes and Wilson 1983). Adenocarcinomas of nasal respiratory epithelium are found in humans, although this is a rare tumor type (Heffner 1983). The histologic appearance is similar to that described in the rat in many cases. Nasal adenocarcinomas in man are associated with occupational exposure to wood and various metal dusts (chromium, nickel, arsenic) (Buiatti et al. 1983; Torjussen 1983).

References

Buiatti E, Geddes M, Carnevale F, Merler E (1983) Nasal cavity and paranasal sinus tumors in woodworkers and shoemakers in Italy compared to other countries. In: Reznik G, Stinson SF (eds) Nasal tumors in animals and man, vol 1. Anatomy, physiology, and epidemiology. CRC, Boca Raton, chap 5

Goodman DG, Ward JM, Squire RA, Chu KC, Linhart MS (1979) Neoplastic and nonneoplastic lesions in aging F344 rats. Toxicol Appl Pharmacol48: 237-248

Hayes HM Jr, Wilson GP (1983) Comparative aspects of nasal passage carcinoma in dogs with man. In: Reznik G, Stinson SF (eds) Nasal tumors in animals and man, vol 2. Tumor pathology. CRC, Boca Raton, chap 10

Heffner DK (1983) Histopathologic classification of human sinonasal tumors. In: Reznik G, Stinson SF (eds) Nasal tumors in animals and man, vol 2. Tumor pathology. CRC, Boca Raton, chap 1

Lee KP, Schneider PW, Trochimowicz HJ (1983) Morphologic expression of glandular differentiation in the epidermoid nasal carcinomas induced by phenylglycidyl ether inhalation. Am J Pathol 111: 140-148

Njoku CO, Chineme CN (1983) Neoplasms of the nasal cavity of cattle and sheep. In: Reznik G, Stinson SF (eds) Nasal tumors in animals and man, vol 2. Tumor pathology. CRC, Boca Raton, chap 8

Patniak AK (1983) Canine and feline nasal and paranasal neoplasms: morphology and origin. In: Reznik G, Stinson SF (eds) Nasal tumors in animals and man, vol 2. Tumor pathology. CRC, Boca Raton, chap 9

Reznik G, Reznik-Schuller H, Ward JM, Stinson SF (1980a) Morphology of nasal-cavity tumours in rats after chronic inhalation of 1,2-dibromo-3-chloropropane. Br J Cancer 42: 772-781

Reznik G, Stinson SF, Ward JM (1980b) Respiratory pathology in rats and mice after inhalation of 1,2-dibromo-3-chloropropane or 1,2,dibromoethane for 13 weeks. Arch Toxicol46: 233-240

Reznik G, Reznik-Schuller HM, Hayden DW, Russfield A, Murthy AS (1981) Morphology of nasal cavity neoplasms in F344 rats after chronic feeding of p-cresidine, an intermediate of dyes and pigments. Anticancer Res 1 : 279-286

Reznik-Schuller HM (1983) Nitrosamine induced nasal cavity carcinogenesis. In: Reznik G, Stinson SF (eds) Nasal tumors in animals and man, vol 3. Experimental nasal carcinogenesis. CRC, Boca Raton, chap 3

72 W. Ellis Giddens Jr. and Roger A. Renne

Schreider JP (1983) Nasal airway anatomy and inhalation deposition in experimental animals and people. In: Reznik G, Stinson SF (eds) Nasal tumors in animals and man, vol 3. Experimental nasal carcinogenesis. eRe, Boca Raton, chap 1

Stinson SF (1983) Nasal cavity cancer in laboratory animal bioassays of environmental compounds. In: Reznik G, Stinson SF (eds) Nasal tumors in animals and man, vol

3. Experimental nasal carcinogenesis. eRe Press, Boca Raton, chap 7

Torjussen W (1983) Nasal cancer in nickel workers. Histopathological findings and nickel concentrations in the nasal mucosa of nickel workers, and a short review of chromium and arsenic. In: Reznik G, Stinson SF (eds) Nasal tumors in animals and man, vol 2. Tumor pathology. eRe, Boca Raton, chap 2

Hemangiosarcoma, Nasal Cavity, Mouse

W. Ellis Giddens Jr. and Roger A. Renne

Synonyms. Hemangioendothelioma.

Gross Appearance

The lesion is not usually visible grossly because it arises in the lamina propria of the nasal mucosa. If, however, invasion occurs through the maxilla to the subcutis, a slight focal bulging of the skin results. When the skin is removed, the underlying subcutis is seen to be dark red.


Hemangiosarcomas arise from the submucosa of the lateral walls of the nasal cavity. The normal pattern ofloose connective tissue and submucosal glands is disrupted by proliferation of endothelial cells with large vesicular or hyperchromatic nuclei. Some of these cells contain mitotic figures. They form small vascular channels and sinusoids (Figs. 107 and 108). These neoplastic channels are often filled with blood, giving the tumor a hemorrhagic appearance. Occasionally, areas of thrombosis, hemorrhage, and necrosis occur in the neoplasms in those portions nearer the lumen of the nasal cavity. As the neoplasms expand, erosion and loss of the overlying respiratory epithelium follows, but the principal direction of invasion is lateral toward the turbinate bones, maxillary sinus, maxilla, marrow space of the maxilla, and the subcutis overlying the maxilla (Fig. 109). These hemangiosarcomas are locally invasive, but there is no evidence of metastasis by way of blood vessels or lymphatics. Transplantation and growth of tumor cells within the respiratory tract has not been observed.


Hemangiosarcomas must be distinguished from hemangiomas and from angiectasis, both of which are also induced by inhalation exposure to propylene oxide. Angiectasis can be distinguished because it is not a neoplasm. The cells are cytologically differentiated and therefore benign. New vascular channels are not formed, but preexisting ones are merely dilated. Hemangiomas are more difficult to distinguish but tend to have larger vascular channels than those in hemangiosarcomas. Mitotic figures are fewer or absent and the endothelial cells are flattened and have a smaller nucleus to cytoplasm ratio. Hemangiomas do not invade the maxilla or maxillary space. A complicating factor in inhalation studies is that rhinitis is often present. In the propylene oxide study to be described, inflammation led to the accumulation of serous or purulent exudate in the lumen of the nasal cavity. The nasal mucosa and submucosa were often heavily infiltrated with lymphocytes, neutrophils, histiocytes, and plasma cells. Fibroplasia and congestion were often observed.

Biologic Features

Hemangiosarcoma of the nasal cavity as a spontaneous event is extremely rare in humans (Bomer and Arnold 1971) and animals. Only one report (Rabstein and Peters 1973) of spontaneous hemangiosarcoma in animals could be found. In this report, only one of 358 BALBI CflCd mice which died or were killed when moribund had a hemangiosarcoma of the ethmoid turbinates.

Fig.t07 (Above). Hemangiosarcoma, submucosa, nasal cavity, mouse. Submucosal glands (G) are disrupted by the proliferation of endothelial cells which form small vascular channels (C). Hemorrhage in nasal cavity (L). A mouse that chronically inhaled 400 ppm propylene oxide. Hand E, x 640 (reduced by 30%)

Experimentally induced hemangiosarcoma in the nasal cavity of mice was observed in a 2-year propylene oxide inhalation study (National Toxicology Program 1984; Renne et aI., to be published). In this study (Table 4),300 B6C3F1 mice were divided into three male and three female groups of 50 each with high-dose (400 ppm), low-dose (200 ppm), and control groups for each sex. Mice were exposed by inhalation 6 h each day, 5 days per week, for 103 weeks.

Hemangiosarcoma, Nasal Cavity, Mouse 73

Fig.t08 (Below). Hemangiosarcoma, submucosa, nasal cavity. Mouse exposed to 400 ppm propylene oxide. Note serous exudate in the lumen (L). Hand E, x 640 (reduced by 30%)

Angiectasis, hemangiomas, and hemangiosarcomas were found at the end of the study in both male and female mice that received the high dose of 400 ppm propylene oxide (Table 4). All except one of the neoplasms were discovered in apparently normal mice that were killed at the end of the study. In this mouse, death occurred as a result of a separate primary neoplasm in another tissue.

74 W. Ellis Giddens Jr. and Roger A. Renne

Fig. 109. Hemangiosarcoma, submucosa of dorsal part of nasal cavity, mouse, following long-term inhalation of 400 ppm propylene oxide. Note the subcutis (S) and invasion ofthe marrow (M) of the maxilla. Hand E, x 160 (reduced by 30%)

Table 4. Nasal lesions in B6C3F1 mice exposed to propylene oxide and in controls'

Male mice Female mice

Oppm 200 ppm 400 ppm o ppm 200 ppm 400 ppm

Angiectasis 0/50 0/50 3/50 0/50 0/50 3/50 Hemangioma 0/50 0/50 5/50 0/50 0/50 3/50 Hemangiosarcoma 0/50 0/50 5/50 0/50 0/50 3/50

• Nasal structure of 50 animals per group was examined microscopically; e.g., five of 50 male high-dose mice had hemangiosarcomas and 13 mice bore lesions


In the mouse study described, hemangiosarcomas of the nasal cavity were locally invasive, extending into submucosal glands, bone, and bone marrow of the maxilla, and subcutis around the maxilla. There was no evidence of vascular or lymphatic spread to other tissues. Primary hemangiosarcomas of the nasal cavity in man, although extremely rare, tend to recur locally after excision. They also have a tendency to metastasize via hematogenous or lymphatic channels to other tissues (Bomer and Arnold 1971).

References

Bomer DS, Arnold GE (1971) Rare tumors of the ear, nose and throat. Acta Otolaryngol [Suppl] (Stockh) 289: 1-25

National Toxicology Program (1984) Inhalation bioassay of propylene oxide for possible carcinogenicity. Terminal report. US Department of Health and Human Services, National Institutes of Health, National Technical Information Service, Springfield, VA

Rabstein LS, Peters RL (1973) Tumors of the kidneys, synovia, exocrine pancreas and nasal cavity in BALB/ cfl

Cd mice. JNCI 51: 999-1006 Renne RA, Giddens WE Jr, Boorman GA, Kovatch R,

Clarke WJ (to be published) Tumors in the nasal cavity of F344 rats and B6CF1 mice induced by inhalation of propylene oxide.

Clear Cell Carcinoma, Larynx, Syrian Hamster 75

Clear Cell Carcinoma, Larynx, Syrian Hamster

Parviz M. Pour

Synonyms. None.

Gross Appearance

Clear cell carcinoma is not usually detected with the naked eye, although the tumor may become large and infiltrate the entire circumference of the larynx (Figs. 110 and 111). In the respiratory tract, the larynx is the usual site, but one lesion has been observed in a male hamster at the bifurcation of the trachea (Pour et al. 1983).


The tumor cells have abundant pale, nearly translucent cytoplasm confined within distinct cell borders, giving the cells an irregular polyhedral shape. Nuclei are large and round to ovoid and contain finely distributed chromatin and occasionally a large nucleolus (Fig. 112). The cells do not stain with periodic acid-Schiff. They grow in solid sheets and bands and have no particular organization (Fig. 111). Since they invade the submucosal glands from outside without penetrating the lumen, pseudoglandular structures may be seen (Figs. 111 and 112). These tumors appear to arise from the basal layer of the respiratory epithelium or of the submucosal glands. They initially appear as single cells or small colonies within the epithelium and grow by expansion and infiltration into the submucosal stroma, elevating the respiratory epithelium. They tend to infiltrate around the submucosal glands and thereby isolate them from the overlying respiratory epithelium (Fig. 112). Metastases to the lungs have been seen (Fig. 113).

Ultrastructure

These lesions have not been studied by electron microscopy.


Inflammatory lesions with intense proliferation of macrophages must be considered in differential

diagnosis as well as certain types of malignant lymphoma. The exact components of the cytoplasm of the cells, when determined, should provide good clues as to the nature of the cells involved.

Biologic Features

Clear cell carcinomas of the larynx are malignant and have a remarkable potential for invasion and early metastases (Fig. 113). Even small tumors may infiltrate blood vessels. The incidence of these lesions has been reported as 1 %-3% (Pour et al. 1976; Pour 1983). The frequency of precursor lesions, which have also been seen in some mutant strains, may be as high as 9% in male hamsters of different colonies (Pour et al. 1979). In animals exposed to certain carcinogens, the frequency of these tumors was not increased, so their development appears to be unrelated to such treatment. Although clear cell carcinoma of the larynx is regarded as a disease of aged hamsters, it has been found in animals only 30 weeks of age. Lesions have been seen more often in males than in females. In an examination of two hamster colonies, the ratio of males to females was 3: 1 (Pour et al. 1976). The etiology of this tumor is unknown.

Comparison with other Other Species

This lesion appears to be limited to the Syrian hamster.

References

Pour PM (1983) Spontaneous respiratory tract tumors in Syrian hamsters. In: Reznik-SchUller HM (ed) Comparative respiratory tract carcinogenesis, vol 1. Spontaneous respiratory tract carcinogenesis. CRC, Boca Raton, chap 7

Pour P, Mohr U, Cardesa A, Althoff J, Kmoch N (1976) Spontaneous tumors and common diseases in two colonies of Syrian hamsters. II. Respiratory tract and digestive system. JNCI 56: 937-948

Pour P, Althoff J, Salmasi SZ, Stepan K (1979) Spontaneous tumors and common diseases in three types of hamsters. JNCI 63: 797-811

76 Parviz M. Pour

Fig.HO (Above). Clear cell carcinoma of larynx with infiltration of the mucosa and submucosa. Hand E, x 26

Fig.Hi (Below). Higher magnification of the same tumor as in Fig. 110, with infiltration of the mucosa and submucosa. Remnants of submucosal glands (arrows) and focal sclerosis around tumor cell nests (lower middle portion). H and E. x 80

Fig. 112 (Above). Clear cell carcinoma oflarynx, surrounding and partially eroding submucosal glands. Hand E, x 390

Clear Cell Carcinoma, Larynx, Syrian Hamster 77

Fig. 113 (Below). Pulmonary metastases of a laryngeal clear cell carcinoma. Hand E, x 195

LESIONS DUE TO INFECTIONS

Murine Respiratory Mycoplasmosis, Upper Respiratory Tract, Rat

Trenton R. Schoeb and 1. Russell Lindsey

Synonyms. Murine chronic respiratory disease; infectious catarrh.

Gross Appearance

Some affected rats have mucopurulent nasal exudate or pink, porphyrin-tinted oculonasal discharge, but gross lesions in the upper respiratory tract are, in many cases, not detectable. Exudate can sometimes be found in the nasal passages, trachea, and tympanic cavities. These structures should be disturbed as little as possible during dissection and collection of specimens for culture so as to preserve the quality of the tissues for microscopic examination.


The principal lesions of murine respiratory mycoplasmosis in the upper respiratory tract are, in decreasing order of frequency, rhinitis, otitis media, laryngitis, and tracheitis. All are characterized by: (a) epithelial changes including hypertrophy, hyperplasia, metaplasia to nonkeratizing squamous or stratified squamous epithelium, and goblet cell hyperplasia; (b) neutrophilic exudation; and (c) accumulation oflymphocytes and plasma cells.

Rhinitis. Normal rat nasal mucosa (Fig. 114) contains few lymphocytes except for small numbers around and just anterior to the nasopharynx. In murine respiratory mycoplasmosis, lymphoid cells accumulate diffusely in the subepithelial stroma. Loss of cilia, pseudo glandular epithelial hyperplasia, and goblet cell hyperplasia can be extensive and severe (Fig. liS).

Otitis Media. The middle ears are nearly as frequently affected as the nasal passages. The tympanic cavity may be completely filled with neutrophils. The lining epithelium, normally simple

squamous or low cuboidal, becomes hyperplastic. Goblet cells are often numerous. In many cases the lumen becomes filled with immature collagenous connective tissue, leaving only a few glandular spaces containing neutrophils at the boundary representing the original lining (Fig. 116). The cavity may eventually clear but the lining membrane remains thickened by connective tissue.

Laryngitis and Tracheitis. The laryngeal submucosal glands are in many cases dilated with mucopurulent exudate. Epithelial changes are as in other tissues, and the tracheal mucosa can become extremely thickened by epithelial hypertrophy and hyperplasia, with formation of gland-like crypts and severe accumulation of lymphoid cells (Figs. 117 and 118).

Ultrastructure

Mycoplasma pulmonis parasitizes the surface of respiratory epithelial cells (Fig. 119). Various degenerative changes occur, ranging from loss of cilia and vacuolation of cytoplasm to necrosis of scattered individual cells. The mechanisms for these changes are unknown, although damage by the accompanying inflammatory response probably contributes.


The characteristic respiratory sounds (snuffling) are not consistently present, but in many cases are the only clinical manifestation of the disease. For unknown reasons, in some affected rats porphyrin secretion from the Harderian glands results in accumulation of red material around the eyes and external nares. Some authors have mistakenly identified this pigment as serosanguinous exudate. In the natural disease, most rats with these signs are infected with M.pulmonis and one or

Murine Respiratory Mycoplasmosis, Upper Respiratory Tract, Rat 79

Fig.114 (Above). Normal nasal septal mucosa. Hand E, x220

Fig.115 (Below). Nasal septal mucosa in severe murine respiratory mycoplasmosis with neutrophilic exudate, flat-

more other agents such as Sendai virus, sialodacryoadenitis virus, rat coronavirus, Streptoco~cus pneumoniae, Corynebacterium kutscheri, Bordetella bronchiseptica, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pasteurella pneumotropica, or Streptobacillus moniliformis. Uncomplicated infections seldom occur. However, M. pulmonis alone is sufficient to produce the full spectrum of lesions of respiratory mycoplasmosis and no other organism has been shown to produce its characteristic lesions in pathogen-free rats. M.pulmonis is therefore the primary pathogen, but clearly other agents do modify the course of the natural disease.

tening of the superficial epithelium, loss of cilia, extensive goblet cell hyperplasia with formation of glandular epithelial infoldings, and diffuse accumulation of lymphocytes and plasma cells. Hand E, x 220

Disease caused by Sendai virus is usually subclinical in adult rats and is characterized by necrotizing bronchiolitis (Jacoby et al. 1979). Sialodacryoadenitis virus and coronavirus do not cause serious respiratory disease in adult rats and do not appear to be important respiratory pathogens, but they can cause focally necrotizing rhinotracheitis and multifocal interstitial pneumonia (Jacoby et al. 1979). These viral lesions, if found, should not be difficult to differentiate from those of respiratory mycoplasmosis. S. pneumoniae and C. kutscheri have been associated with rhinitis and otitis media, although in most cases M. pulmonis is probably also present. Other bacteria are

80 Trenton R.Schoeb and J.RusseU Lindsey

Fig. 116. Tympanic bulla with purulent exudate in lumen (AJ and fibrosis of the lining membrane. Hand E, x 35

probably little more than opportunistic pathogens. Diagnosticians must diligently gather all information necessary to identify all agents present in affected rats. Results of histological studies, bacterial and mycoplasmal cultures, and serological tests must be carefully considered in making diagnoses. A diagnosis of murine respiratory mycoplasmosis can be supported by enzyme-linked immunosorbent assay (ELISA) of serum antibodies (Horowitz and Cassell 1978) and by cultural recovery of the organism. Failure to isolate the organism does not rule out the diagnosis, as the organism can be quite difficult to grow by routine culture methods. Among the difficulties which can be encountered is growth inhibition by certain tissue and medium components (Del Giudice et al. 1980; Kaklamanis et al. 1971; Mardh and Taylor-Robinson 1973; Tauraso 1967). Davidson et al. (1981) have reported that cultural isolation and ELISA both detected a high percentage of infected rats, and that combinations of methods increased the detection rate. Culturing multiple sites in the respiratory tract also increased the rate of recovery of organisms, but of individual sites the organism was most frequently isolated from the nasopharyngeal duct.

Biologic Features

Several aspects of the natural history of murine respiratory mycoplasmosis need to be clarified. The major mode of transmission is probably via aerosol from affected mothers to neonates, but in utero transmission apparently occurs also. Infection results in a slowly progressive respiratory disease which persists throughout the animal's life. Infected rats can transmit the infection to others but horizontal transmission is slow, even within a cage, and is considerably reduced by increasing the space between cages. Transmission of M.pulmonis via food, water, bedding, and other materials has been suggested but not proved. Inasmuch as M. pulmonis has been isolated from wild rats, cotton rats, rabbits, Syrian hamsters, and guinea pigs, these animals could be potential sources of infection. In conventional and experimentally infected rats, the nasal passages and middle ears are the most commonly infected sites; lung lesions are less consistently found. Thus the upper respiratory tract seems to be the source of infection for the distal tract. The extent to which the distal airways and lungs become affected seems to depend on complex interactions among host, organism, and environment. Exposure to ammonia from soiled cage bedding or to purified ammonia increases the severity of upper respiratory lesions and both


Fig. 117 (Above). Normal rat trachea. Hand E, x 330

Fig. 118 (Below). Tracheal mucosa in severe chronic murine respiratory mycoplasmosis, with neutrophilic exudate, epithelial hyperplasia, loss of cilia, and mucosal thickening

the incidence and severity of lung lesions (Broderson et al. 1976). The mechanisms of this effect remain unclear, but ammonia greatly increases the growth of M. pulmonis in rat respiratory tracts, particularly in the nasal passages, probably through effects on the host rather than on the organism itself (Schoeb et al. 1982).

with accumulation of many lymphocytes and plasma cells. The dark line at the epithelial surface represents numerous mycoplasmas adherent to the cells (see Fig. 119). Hand E, x 330

Other microbial agents are frequently found in colonies and it is likely that some of them can affect the expression of mycoplasmosis. Sendai virus is a likely contributor because, although no such studies in rats have been reported, Sendai virus infection in mice enhances intrapulmonary growth of M.pulmonis (Howard et al. 1978), and

82 Trenton R. Schoeb and 1. Russell Lindsey

Fig. 119. Tracheal epithelial cell with numerous M.pulmonis organisms on its surface. TEM, x 12000

alters functions of alveolar macrophages and inhibits pulmonary bacterial clearance (Jakab 1981). Increased susceptibility is also associated with advancing age, possibly as a result of decreased immune responsiveness (Cassell et al. 1979). Genetically determined factors are also important inasmuch as LEW rats are more susceptible than F344 rats (Davis and Cassell 1982; Davis et al. 1982). Mycoplasma pulmonis inhabits the surface of ciliated epithelial cells, as do other mycoplasmas affecting the respiratory tract (Cassell et al. 1978). This relationship is undoubtedly fundamental to the initiation and maintenance of infection. For example, it may enable the organism to escape elimination by the mucociliary system, cellular and noncellular inflammatory processes, and specific immune effector mechanisms (Cassell et al. 1978). It seems likely that the nonspecific mitogenic activity of M. pulmonis (Naot et al. 1979) alters lymphocyte responsiveness and misdirects or disrupts specific immune responses (Cassell et al. 1979). How M.pulmonis damages epithelial cells is unknown, but it probably competes for essential metabolites or cell components. Production of toxic wastes has been suggested but not proved to be associated with pathogenicity. Mycoplasma pulmonis infection is ubiquitous in conventional rat colonies. Studies have shown by ELISA and cultural isolation that infection is also common in "barrier-maintained" colonies in the United States and Great Britain (Cassell et al.

1981). Rats from these colonies had mild or no lesions, not the classical lesions described here. The organism has also been found in rats thought to be germ-free (Ganaway et al. 1973).


With the exception of contagious pleuropneumonia (Mycoplasma mycoides) of cattle and goats, which is characterized by fibrinous pleuropneumonia, most respiratory mycoplasmoses are morphologically similar. In mice, lesions of murine respiratory mycoplasmosis are similar to those in rats, with a few minor differences. Middle ear fibrosis like that seen in rats does not occur in mice. Lymphoid accumulations in mice contain a greater proportion of plasma cells, as do the regional lymph nodes. Syncytial giant cells in the epithelium of the nasal passages occur in mice with the disease but not in rats. Lesions of Mycoplasma gallisepticum infection in chickens include chronic suppurative rhinitis, sinusitis, tracheitis and bronchitis with epithelial hypertrophy and hyperplasia, increased mucosal mucus production, and lymphoid cell accumulation with follicle formation in the lamina propria. The lesions are thus similar to those of the murine respiratory disease. M. gallisepticum alone usually causes a rather mild upper respiratory disease, but infection is frequently complicated by wild or vaccine Newcastle disease virus, infectious bronchitis


virus, avian adenovirus, or Escherichia coli, resulting in more severe disease with extension to the lungs and air sacs. In turkeys, M. gallisepticum produces a disease similar to that in chickens but with even more of a tendency for upper respiratory tract lesions, especially sinusitis, to predominate. Thus the disease is usually called infectious sinusitis. M. meleagridis causes a spontaneously resolving air sacculitis also characterized by chronic suppurative inflammation, lymphoid infiltration, and epithelial hyperplasia. Swine infected with Mycoplasma hyopneumoniae develop lesions similar to those of murine respiratory mycoplasmosis, although bronchiectasis almost never occurs. Pneumonic lesions with macrophage and neutrophil accumulations are more prominent than in the rodent disease, and the gross lesions, discrete gray-red firm masses predominantly in the dependent parts of the lungs, are characteristic of porcine "enzootic pneumonia." The natural disease is frequently complicated by other agents such as Pasteurella multocida, Mycoplasma hyorhinis, and swine adenovirus. Lesions of M. pneumoniae infection in humans are not well known because the disease is rarely fatal. However, available descriptions indicate that lesions include peribronchial and perivascular lymphoid infiltrates, acute bronchitis and bronchiolitis, transformation of alveolar epithelium to cuboidal type and an alveolar exudate made up chiefly of macrophages. These changes are similar to those of other respiratory mycoplasmoses.

References

Broderson JR, Lindsey JR, Crawford JE (1976) The role of environmental ammonia in respiratory mycoplasmosis of rats. Am J Pathol 85: 115-130

Cassell GH, Davis JI(, Wilborn WH, Wise KS (1978) Pathobiology of mycoplasmas. In: Schlessinger D (ed) Microbiology 1978. American Society for Microbiology, Washington DC, pp 399-403

Cassell GH, Lindsey JR, Baker HJ, Davis JK (1979) Mycoplasmal and rickettsial diseases. In: Baker HJ, Lindsey JR, Weisbroth SH (eds) The laboratory rat, volt. Academic, New York, chap 10

Cassell OH, Lindsey JR, Davis JK, Davidson MK, Brown MB, Mayo JO (1981) Detection of natural Mycoplasma pulmonis infection in rats and mice by an enzyme linked immunosorbent assay (ELISA). Lab Anim Sci 31: 676-682

Davidson MK, Lindsey JR, Brown MB, Schoeb TR, Cassell GH (1981) Comparison of methods for detection of Mycoplasma pulmonis in experimentally and naturally infected rats. J Clin Microbiol14: 646-655

Davis JI(, Cassell GH (1982) Murine respiratory mycoplasmosis in LEW and F344 rats: strain differences in lesion severity. Vet Pathol19: 280-293

Davis JI(, Thorp RB, Maddox PA, Brown MB, Cassell GH (1982) Murine respiratory mycoplasmosis in F344 and LEW rats: evolution of lesions and lung lymphoid cell populations. Infect Immun 36: 720-729

DeI Giudice RA, Gardella RS, Hopps HE (1980) Cultivation of formerly noncultivable strains of Mycoplasma hyorhinis. CUff Microbiol4: 75-80

Ganaway JR, Allen AM, Moore TD, Bohner HJ (1973) Natural infection of germ-free rats with Mycoplasma pulmonis. J Infect Dis 127: 529-537

Horowitz SA, Cassell GH (1978) Detection of antibodies to Mycoplasma pulmonis by an enzyme linked immunosorbent assay. Infect Immun 22: 161-170

Howard CJ, Stott EJ, Taylor G (1978) The effect of pneumonia induced in mice with Mycoplasma pulmonis on resistance to subsequent bacterial infection and the effect of a respiratory infection with Sendai virus on the resistance of mice to Mycoplasma pulmonis. Gen Microbioi 109: 79-87

Jacoby RO, Bhatt PN, Jonas AM (1979) Viral diseases. In: Baker HJ, Lindsey JR, Weisbroth SH (eds) The laboratory rat, vol 1. Academic, New York, chap 11

Jakab GJ (1981) Interactions between Sendai virus and bacterial pathogens in the murine lung: a review. Lab Anim Sci 31: 170-177

Kaklamanis E, Stavropoulos I(, Thomas L (1971) The mycoplasmacidal action of homogenates of normal tissues. In: Madoff S (ed) Mycoplasma and the L-forms of bacteria. Gordon and Breach, New York, pp 27-35

Mardh PA, Taylor·Robinson D (1973) New approaches to the isolation of mycoplasmas. Med Mikrobiol Immunol (Berl) 158:259-266

Naot Y, Merchav S, Ben-David E, Ginsburg H (1979) Mitogenic activity of Mycoplasma pulmonis. I. Stimulation of rat Band T lymphocytes. Immunology 36: 399-406

Schoeb TR, Davidson MI(, Lindsey JR (1982) Intracage ammonia promotes growth of Mycoplasma pulmonis in the respiratory tract of rats. Infect Immun 38: 212-217

Tauraso NM (1967) Effect of diethylaminoethyl dextran on the growth of mycoplasma in agar. J Bacteriol 93: 1559-1564

84 David G. Brownstein

Sialodacryoadenitis Virus Infection, Upper Respiratory Tract, Rat

David G. Brownstein

Synonyms. Rat submaxillary gland virus infection; SDAV infection.

Gross Appearance

Gross lesions are usually extrarespiratory and limited to mixed or serous salivary glands, exorbital glands, Harderian glands, peri glandular connective tissue, cervical lymph nodes, and thymus. The submaxillary and parotid salivary glands are enlarged, pale, and edematous. Intermandibular and cervical connective tissue is gelatinous due to periglandular edema. This edema restricts the venous return in the neck, resulting in distention of the great veins entering the thoracic inlet. Exorbital glands are occasionally enlarged. Harderian glands are swollen and flecked with yellow-gray spots. These foci must be distinguished from normal brown-red mottling of the Harderian gland imparted by its normal content of porphyrin pig-

ment. The cervical lymph nodes are enlarged and the thymus is atrophic. In these cases, ocular lesions may include corneal opacity, corneal ulcers, pannus, hypopyon, hyphema, and megaloglobus (Innes and Stanton 1961; Jacoby et al. 1975, 1979).


Respiratory lesions are primarily restricted to the upper respiratory tract. They precede inflammatory changes in the exocrine tissues of the head. Over the course of approximately 5 days, beginning on the 2nd day of infection, there is spreading necrosis of respiratory epithelium in the nasal cavity accompanied by congestion, edema, and mixed inflammatory infiltrate of the lamina propria (Figs. 120 and 121). The epithelial lining of the turbinates is most severely affected; olfactory epithelium is usually spared. Some meatuses are

Fig. 120. Ventral turbinate and lateral wall of nasal cavity in rat experimentally infected with sialodacryoadenitis virus. Note exudative inflammation of the mucosa with gaps in epithelial integrity. H and E, x 240 (reduced by 15%)

Sialodacryoadenitis Virus Infection, Upper Respiratory Tract, Rat 85

covered by exudate composed of necrotic epithelium, neutrophils, and mucus. Despite a tropism of this virus for serous or mixed salivary glands, the serous mucosal glands of the nasopharynx sustain relatively mild injury. Necrotic ducts and acini within mucosal glands do occur, however, and afford some specificity to the lesion. There is qualitatively similar inflammation in the trachea, but changes are milder and less uniform than in the nasopharynx. Upper respiratory lesions are resolved by the end of the 2nd week of infection. Lung changes are confined to mild hyperplasia within peribronchial lymphoid nodules (Jacoby et al. 1975, 1979). Severe inflammatory changes occur within mixed or serous salivary glands, exorbital glands, and Harderian glands. Description of these changes is beyond the scope of this volume. The reader is referred to several excellent studies of sequential changes in these tissues (Innes and Stanton 1961; Jacoby et al. 1975, 1979).

Ultrastructure

We have found no report of the ultrastructural features of respiratory tract lesions caused by SDA V, but these features have been studied in infected submaxillary gland epithelium (Jonas et al. 1969). Infected epithelial cells have focally dilated cisternae of endoplasmic reticulum and cytoplasmic vesicles which contain spherical dense or hollow cores, 60-70 nm in diameter, surrounded by an envelope 80-120 nm in diameter. The characteristic corona, seen in negatively stained preparations, is not seen by transmission ultramicroscopy. Morphologically, sialodacryoadenitis virus is indistinguishable from Parker's rat coronavirus.


Upper respiratory tract lesions must be distinguished from those caused by Parker's rat coronavirus, Sendai virus, pneumonia virus, Mycoplasma pulmonis, and pathogenic bacteria. Pneumonic changes, which frequently accompany rhinitis caused by Parker's rat coronavirus, Sendai virus, and pneumonia virus, have not been reported in SDAV infection. A careful histopathological examination of the exocrine tissues of the head is usually sufficient to enable one to provisionally diagnose SDA V infection, but rhinotracheitis can precede changes in these tissues.

Fig. 121. Ventral turbinate of a rat experimentally infected with sialodacryoadenitis virus. Much of the epithelium is necrotic and desquamated. Some leukocytes are present in the lumen. Hand E, x 740 (reduced by 15%)

Biologic Features

Natural History. Sialodacryoadenitis virus causes acute limited infections; there is no evidence for a carrier state. The virus is highly contagious and is transmitted by aerosol, direct contact, and fomites. There are two patterns of infection. Enzootic infections occur primarily in breeding colonies, where sucklings are passively immune, adults are actively immune, and weanlings are a continuous source of susceptible individuals due to waning passive immunity. It is therefore weanlings that generally exhibit clinical signs. Explosive epizootics occur in non immune colonies with the highest morbidity in weanlings. Signs are usually transient and consist of intermandibular and cervical edema, swelling of submaxillary glands, sneezing, nasal and ocular discharges which are often red-tinged due to a high content of porphyrin, photophobia, and keratoconjunctivitis and its sequelae. Some complications of keratoconjunctivitis, such as glaucoma and phthisis, cause permanent disfigurement (Jacoby et al.


1979). Subclinical infections are common. Extensive host range studies have not been done but SDA V can experimentally infect mice by the respiratory route (Bhatt et al. 1977).

Pathogenesis. Sialodacryoadenitis virus is epitheliotropic, with replication limited to the respiratory tract and certain exocrine tissues of the head and neck. It replicates at all levels of the respiratory tract but produces disease primarily in the upper respiratory tract, where the highest titers are achieved. Virus is excreted for 7 days, after which it is cleared and neutralizing and complement-fixing antibodies appear in the serum (Jacoby et al. 1975, 1979).

Etiology. Sialodacryoadenitis virus (Coronaviridae) is a pleomorphic, enveloped RNA virus with plump. pedunculated surface projections (corona). It is approximately 114nm in diameter. The virus replicates intracytoplasmically and virions are formed in cytoplasmic vesicles and endoplasmic reticulum (Jacoby et al. 1979). The virus is closely related antigenically to Parker's rat coronavirus (Bhatt et al. 1972).

Frequency. Coronavirus infections are common in commercial and institutional rat colonies (Jacoby et al. 1979; Parker et al. 1970). Because of the close antigenic relationship of SDAV to Parker's rat coronavirus, seroconversion to both viruses occurs in SDAV-infected rats. It is therefore difficult to confirm SDAV infection by serology alone (Bhatt et al. 1972; Jacoby et al. 1979).


Coronaviruses are ubiquitous in humans, animals, and birds (Bohl 1981). Although coronaviruses cause respiratory infections in chickens, humans, and rats, SDAV (and to a limited degree Parker's rat coronavirus) is the only coronarvirus that replicates and produces disease in salivary, exorbital, and Harderian glands.

References

Bhatt PN, Percy DH, Jonas AM (1972) Characterization of the virus of sialodacryoadenitis of rats: a member of the coronavirus group. J Infect Dis 126: 123-130

Bhatt PN, Jacoby RO, Jonas AM (1977) Respiratory infection in mice with sialodacryoadenitis virus, a coronavirus of rats. Infect Immun 18: 823-827

Bohl EH (1981) Coronaviruses: diagnosis of infections. In: Kurstak E, Kurstak C (eds) Comparative diagnosis of viral diseases, vol 4. Academic, New York, chap 7

Innes JRM, Stanton MF (1961) Acute diseases of the submaxillary and Harderian glands (sialodacryoadenitis) of rats with cytomegaly and no inclusion bodies. Am J Pathol 38: 455-468

Jacoby RO, Bhatt PN, Jonas AM (1975) Pathogenesis of sialodacryoadenitis in gnotobiotic rats. Vet Pathol 12: 196-209

Jacoby RO, Bhatt PN, Jonas AM (1979) Viral diseases. In: Baker HJ, Lindsey JR, Weisbroth SH (eds) The laboratory rat, vol 1. Academic, New York, chapt 11

Jonas AM, Craft J, Black CL, Bhatt PN, Hilding D (1969) Sialodacryoadenitis in the rat. A light and electron microscopic study. Arch Pathol88: 613-622

Parker JC, Cross SS, Rowe WP (1970) Rat coronavirus (RCV): a prevalent, naturally occurring pneumotropic virus of rats. Arch Virusforsch 31: 293-302

The Lung (Bronchi, Bronchioles, Alveolar Ducts, Alveoli, Pleura)

HISTOLOGY AND ULTRASTRUCTURE

Structure and Function of the Lung

Charles Kuhn III

This paper will assume that the outlines of lung structure are known by the reader and will emphasize some of the newer information that has accumulated in the last few years about the biology of various types of cells which are found in the lung and airways. It will be convenient to discuss the airways, where bulk flow of air occurs, separately from the acini, where gas exchange with the circulation takes place.

Conducting Airways

The airways consist of a bifurcating series of muscular tubes of considerable complexity. The number ofbranchings varies in different regions of the human lung from eight to perhaps 25 generations before acini are reached. The larger airways are reinforced with cartilage and are called bronchi; airways without cartilage are bronchioles. In the common laboratory rodents, rats, mice, and hamsters, the only bronchi are extrapulmonary; all airways within the lungs lack cartilage. In large

Fig. 122. Epithelium of rat trachea. Nuclei of the basal cells (a) are darkly stained and oriented horizontally. The predominant columnar cells are ciliated and only scattered

animals, including man, the larger intrapulmonary airways also contain cartilage. In the extrapulmonary airways, the cartilage is in the form of a horseshoe-shaped incomplete ring, whereas in intrapulmonary airways of the larger animals it is present as discontinuous islands of cartilage, several at a given level. It is this difference in organization which enables us to cough (Horsfield 1974). When one coughs, one exhales initially against the closed glottis and generates a considerable amount of positive intrathoracic pressure. This pressure would collapse the extrapulmonary bronchi and trachea if they were not reinforced by the horseshoe-shaped cartilage. The intrapulmonary airways, however, are tethered by the surrounding lung parenchyma so that they will not collapse. However, during the early phase of coughing there is muscular constriction of the bronchi which markedly narrows the lumen. Although the discrete islands of cartilage provide some stiffening of the airway walls, they still permit them to narrow. Then when the glottis opens, the narrowing of airway lumina results in the very

secretory cells are seen (b). Epoxy section, toluidine bluebasic fuchsin, x 1000

90 Charles Kuhn III

Fig.123. Serous cell of rat bronchial surface epithelium. The secretory granules are electron dense and discrete. Electron micrograph, x 14000

high rate of flow necessary to dislodge material within the airway. The epithelium which lines the airways is a pseudostratified ciliated columnar epithelium consisting of basal cells, ciliated cells, and secretory cells, all resting on the basal lamina with nuclei at different levels (Fig. 122). The basal cells, which do not cause the airway lumen to contract, are the cells which label most heavily with tritiated thymidine and are presumed to serve as stem cells for the columnar cells. Ciliated and secretory cells are concerned with the production and propulsion of the mucous blanket which provides the mechanical protection of the airways against organisms and dusts deposited from the inhaled air. The mucous blanket is conceived as consisting of two phases: a surface layer which consists of mucus and an underlying layer of watery fluid in which the cilia beat. The source of watery fluid in this underlying layer is not known with any certainty. No specific cell type has been associated with its production. Its thickness is probably regulated by the transport of ions across the airway epithelium, since physiological studies have shown that tracheal epithelium can transport ions

(Nadel et al. 1979). There is a chloride flux from the interstitial space to the bronchial lumen and both a sodium and a chloride flux from the lumen to the interstitium. These ion fluxes will passively carry water osmotically, and this seems a reasonable mechanism to control the thickness of the serous layer. The mucous blanket is a continuous layer. The former controversy about whether it consisted of patches or a continuous coating (van As 1977) was resolved by fixing the tissues by perfusion through the vascular system, which precipitates the mucus without dislodging it, revealing the continuous lining (Luchtel1978). The sources of the mucus are twofold - the glands and the surface epithelium. The surface epithelium has two types of secretory cells (Jeffery and Reid 1975). The familiar goblet cell has an electron-opaque cytoplasm with a considerable amount of endoplasmic reticulum and has large secretory vesicles with a very pale lucent content. The vesicles are often fused with one another and secretion into the bronchial lumen takes place by compound exocytosis. The other type of secretory cell in rodents is the serous cell of the bronchial surface epithelium (Fig. 123). The cell has a relatively electron-lucent cytoplasm but also has an abundance of endoplasmic reticulum. Its secretory granules are discrete and electron dense and do not fuse with one another. The other source of mucus secretion is the mucous glands, which in laboratory rodents such as the hamster or rat are found only in the trachea, but in larger animals such as dog, cat, or man extend as far as the cartilage does into the peripheral bronchi (Spicer et al. 1971). The mucous glands are compound tubular acinar glands. The more central portions of the tubules are lined by cells distended with mucus -the more peripheral parts are lined by serous cells with large apical eosinophilic granules (Fig. 124). By electron microscopy, the distended or mucous cells have relatively electron-lucent secretory vesicles and scant, relatively electron-dense cytoplasm containing endoplasmic reticulum. The serous cell of the tracheobronchial glands is characterized by a large amount of lamellar endoplasmic reticulum which occupies the basal portion of the cell. The apical portion of the cell contains O.5-2Ilm discrete electron-dense secretory granules. These granules contain either neutral or acidic glycoproteins. In addition to producing the glycoproteins to be added to the bulk phase of the mucous blanket, serous cells produce specific substances which are probably important in defense against infectious

Fig.124. Seromucous glands from rat trachea. Note large cytoplasmic granules in the serous cells clustered at the ends of secretory tubules (a). On the left, a plasma cells (b)

agents. By immunoperoxidase staining, the granules contain lysozyme (Bowes and Corrin 1977), an enzyme which is active in the hydrolysis of components of certain gram-positive bacterial cell walls, and lactoferrin, an iron-containing protein whose function as an antibacterial agent is not understood. In addition, the serous granules contain a protease inhibitor (Mooren et al. 1982) with a low molecular weight of about 15000 daltons, which inhibits the activity of the leukocyte-derived proteases cathepsin G and elastase. As a consequence, this protease inhibitor has been called "antileukoprotease." Its importance lies in the fact that during infection the bronchial mucus becomes purulent and leukocytes degenerate, die, and release potentially injurious proteases. Presumably, the antileukoprotease prevents damage to the cells lining the airways by inactivating the proteases. The third cell in the mucous glands, the myoepithelial cell, is represented in the light microscope by elongated nuclei flattened against the basal lamina. By electron microscopy these cells resemble smooth muscle; their cytoplasm is filled with contractile filaments with dense attachment bodies. However, the cells lie within the basal lamina and form desmosomal junctions with

Structure and Function of the Lung 91

lies in proximity to the gland. Epoxy section, toluidine blue, x 1000

neighboring mucous and serous secretory cells. Although it is properly an epithelial cell, its ultrastructural features indicate that it is specialized for contraction and allow the inference that its contraction helps to express the mucous secretions into the bronchial lumen. Plasma cells are also associated with the mucous glands (Fig. 124). The main immunoglobulin of the mucous secretions is secretory IgA. About 50% of the plasma cells around the mucous glands contain IgA. Both the mucous and serous cells contain a secretory piece in their plasma membrane. The assumption is that the secretory IgA is produced in the plasma cells and is transported across the membrane of secretory cells and into the glandular lumen. The mucous blanket is propelled by the action of the ciliated cells. There are approximately 200 cilia per cell arising from basal bodies in the apex of the cell. Beneath the basal bodies, mitochondria are concentrated in the apical cytoplasm to supply the adenosine triphosphate (ATP) required for ciliary beating. The active machinery of the cilia is known as the axoneme (Gibbons 1981). The axoneme consists of nine peripheral doublet pairs of microtubules arranged around the periphery of two central single microtubules

92 Charles Kuhn III

---D

r--~--O

i;;;jjjj ...... ~-R

:.;;;....;;:---- C

(Fig. 125). Attached to each of the doublets at the periphery are a number of accessory structures. Paired arms are attached to one of the tubules of each doublet (the A subfiber). These projecting arms contain an ATPase, dynein, which is involved in transduction of the energy from ATP for ciliary beating. Radially arranged structures which extend from the A subfibers toward the central pair are termed "radial spokes." Connecting each A subfiber to the B subfiber of the adjacent doublet is a very fine thread called the "nexin link," which is difficult to see in most electron micrographs. The C-shaped projections attached to the central singlet microtubules are the central sheath. When the cilium beats, it requires magnesium ions and A TP. In the presence of magnesium the dynein arms form connections to specific sites on the B subfiber of the adjacent doublet. As ATP is hydrolyzed they detach again, but during the detachment they presumably undergo conformational changes and rotate. This rotation produces sliding between the adjacent peripheral doublets (Warner 1978). The sliding is transformed into a bend because the sliding shear is restrained by the presence of the radial spokes which hook up to the central sheath and by the nexin links. This causes the cilium to bend rather than to shoot apart. If trypsin is used to digest the nexin links and the radial spokes, exposure of cilia to A TP causes the cilia to extrude the doublet tubules rather than bend (Lindemann and Gibbons 1975). Why on earth does anybody in veterinary or human medicine want to know so much detail about how cilia work? It turns out that there are subjects

Fig. 125. Cross section of human respiratory cilium. Peripheral microtubular doublet (D), outer dynein arm (0), inner dynein arm (I), radial spoke (R), central singlet microtubule (C). Electron micrograph, x 200000

in whom cilia fail to beat, producing a condition called the "immotile cilia syndrome" (Mzelius 1981), which occurs in man and has also been described in the dog. Subjects with immotile cilia have repeated bronchial, sinus, and middle ear infections beginning soon after birth. In one particular series from the Sick Children's Hospital in Toronto, about a third of the patients had bronchiectasis (Corkey et al. 1981). In adults, the proportion of patients with bronchiectasis is probably higher. Some patients develop nasal polyps and digital clubbing; males are almost invariably sterile. Half the affected subjects have situs inversus. Situs inversus combined with the other features of immotile cilia syndrome is called "Kartagener's syndrome". A variety of structural abnormalities of the cilia give rise to the immotile cilia syndrome. The most common abnormality is absence of the dynein arms. Other abnormalities which have been described include absence of the radial spokes rather than absence of the dynein arm, selective deficiencies of either the outer dynein arm or inner dynein arm alone, and transposition of a microtubular doublet. Within any given family the abnormalities breed true, so presumably there are different genetic abnormalities (Mzelius 1981). Those who are involved in evaluating inhalation toxicology using electron microscopy may come across a variety of abnormal forms of cilia. Many of these are nonspecific abnormalities and do not connote a herditary problem. Chronic irritation to the bronchi can lead to abnormal ciliary structures such as compound cilia, absence of the cen-

Fig. 126. Endocrine-type cells (E) in a neuroepithelial body, hamster lung. Cytoplasm is filled with dense core-type granules. Notice unmyelinated nerve (N) beneath the capillary (C). Electron micrograph, x 12000 (reduced by 15%)

tral tubules or abnormal central tubules, and partialloss of dynein arms. Any abnormality which involves only a small fraction of the cilia is probably an acquired abnormality and not hereditary. Not all the cells present in the airways can be recognized in a hematoxylin and eosin section. Rare cells with well-developed microvilli like those of a brush border (brush cells) can be identified by electron microscopy in airways from trachea to alveolus (Hijiya 1978). Granulated cells can be seen with the aid of silver stains or by using ultraviolet light fluorescence or electron microscopy (Fig. 126). These cells are called "small granule cells," "endocrine-like cells," or sometimes "Kultschitzky cells" (Bensch et al. 1965). They contain numerous small granules, 0.2-0.4 ~m in diameter, and occur in two forms. Some are discrete cells which occur at all levels of the tracheal bronchial tree, in small airways or large airways; in fact, small numbers of them can even be found in the mucous glands. Others are organized corpuscular collections of argyrophilic cells, which contain both afferent and efferent nerves. The capillaries in the underlying bronchial mucosa associated with these structures have a fenestrated type of endothelium. These specialized structures have been identified by Lauweryns as neuroepithelial bodies.


Neuroepithelial bodies occur preferentially near the points of bifurcation of airways. Lauweryns et al. (1977, 1978) have presented evidence that these bodies function as chemoreceptors which are sensitive to the oxygen tension in the inspired air. Animals exposed to hypoxic atmospheres undergo vasoconstriction. Lauweryns' theory is that the stimulation of these organized chemoreceptive structures in the airways mediates hypoxic vasoconstriction. The evidence for this is that cells in the neuroepithelial bodies degranulate when animals are exposed to hypoxia. Within the last few years much has been learned about the contents of those granules. The specific substances identified include serotonin and several peptide hormones identified by immunohistochemistry, including calcitonin and the neuropeptides bombesin and enkephalin (Cutz et al. 1981). The function of these peptides is quite unknown at the present time, but they do serve as potential markers for identifying tumors arising from this cell type. In fact, bombesin seems to be uniformly present in the human bronchial tumor called oat cell carcinoma (Erisman et al. 1982). As one moves from the cartilage-containing-Iayer airways into the bronchioles, there is a shift in the type of epithelium encountered. Ciliated cells still persist but other secretory cells are found: the

94 Charles Kuhn III

Clara cells or nonciliated bronchiolar cells (Fig. 127). The ultrastructural features of the Clara cells are three: they have a conspicuous protruding dome-shaped apex, secretory granules (which in the rat can be either generally spherical or rodshaped), and a very extensive smooth endoplasmic reticulum (in most rodents). The granules in Clara cells do not contain mucin. In some species they are PAS positive, in other species they are not. The granules are digestible by pepsin, which is the best available evidence that they contain a basic protein. The granules appear to be secreted by conventional exocytosis to the bronchiolar surface, where they contribute to the bronchiolar lining layer (Kuhn et al. 1974; Ebert et al. 1976). In addition to their secretory function, Clara cells are also important as stem cells in the repair of bronchiolar injury. The ciliated cells are particularly susceptible to injury by inhaled toxins, but have little or no capacity to divide, so that the Clara cells are the source of most repair in bronchiolar injury. One might predict that the Clara cells of rodents, with their extensive smooth endoplasmic reticulum, would have the enzymatic machinery for metabolizing simple organic chemi-

cals. In fact, Clara cells have even higher levels of cytochrome p4so than do the hepatocytes (Serabjit-Singh et al. 1980), and are rather selectively injured by number of simple compounds such as carbon tetrachloride, naphthalene, and 4-ipomeanol (Boyd et al. 1980). It is uncertain at the present time, however, whether the results of experiments on rodents can be applied to other species, since in many species, including primates, the smooth endoplasmic reticulum is not well developed. The only endoplasmic reticulum in the Clara cells in human bronchioles, for example, is rough endoplasmic reticulum. In some rodent species lymphoid nodules scattered along the intrapulmonary bronchi are called "bronchus-associated lymphoid tissue," or BALT. BAL T is well developed in rabbits and rats but is absent in the hamster and, probably, in man (McDermott et al. 1982). BALT is a lymphoepithelial structure rather similar to the palatine tonsil. In addition to aggregates of lymphocytes with germinal centers in the submucosa, lymphocytes actually appear in the epithelium of the mucosa. The epithelium overlying the BAL T is nonciliated and seems to be unusually permeable to antigens,

Fig. 127. Bronchiole, rat lung. Brush cell (B). Clara cell (C), ciliated cell (Ci), macrophage (M). Macrophages can be seen on airways as well as alveolar surfaces. Electron micrograph, x 6000

so inhaled antigens may be preferentially transported across this special epithelium to reach the lymphoid tissue. In man there does not seem to be much BAL T, but lympoid nodules do occur near the bifurcations of respiratory and membranous bronchioles and are associated with the beginning of the lymphatic system.

Acini

The gas-exchanging units of the lung are termed "acini" (Weibel 1973). Each acinus is the unit of tissue supplied by a terminal bronchiole and consists of respiratory bronchioles, alveolar ducts, alveolar sacs, and their associated alveoli (Fig. 127). In rodents, respiratory bronchioles are almost nonexistent: at most a single short respiratory bronchiole is present. In man, acini have from two to five and usually three generations of respiratory bronchioles. The pulmonary arteries accompany the bronchioles and enter the acini with the terminal bronchioles (Fig. 127). The arterioles accompany the alveolar ducts, giving off capillaries as lateral branches which form a closely woven network in the alveolar walls before draining into the veins at the periphery of the acini. Each vein receives oxygenated blood from two or

Fig.128. Peripheral lung tissue, hamster lung. Bronchiole (B), artery (A), alveolar duct (AD). The artery accompanies the bronchiole and extends along the alveolar duct. The


three acini. Thus the arteries accompany the bronchi while the veins drain separately. Lymphatics start around the small arteries and veins and drain centrally. No lymphatics actually extend out into the alveolar septa. The walls of the distal airspaces have a similar structure whether these are alveoli or alveolar ducts (Fig. 128). They are covered by a thin epithelium and consist mainly of capillaries arranged in a grid-like anastomosing pattern best appreciated by focusing up and down on sections much thicker than the usual diagnostic sections, approximately 50!lm thick. The capillaries are lined by the nonfenestrated type of endothelial cells, joined by tight junctions which are moderately permeable to macromolecules if compared to the epithelium, which has very "tight" junctions (Inoue et al. 1976). The airspace walls are strengthened by connective tissue bundles containing collagenous and elastic fibers. The connective tissue bundles weave back and forth through the capillary grid, but in any given section through the airspace wall the connective tissue space lies to one side of the capillary. On the opposite side the capillary is separated from the airspace only by a thin layer of epithelium with which it shares a common basal lamina (Fig. 128). Should fluid filtration through the capillaries increase for any

lung was fixed by perfusion at approximately functional residual capacity. SEM, x 100 (reduced by 15%)

96 Charles Kuhn III

Fig.129 (Above). Cross section through interalveolar septum of hamster lung with two adjacent alveoli (A). The capillary (C) is separated from the alveolus below by a very delicate barrier consisting only of attenuated endothelium, epithelium, and shared basal lamina. A type I epithelial cell in the center is sectioned through its nucleus (/). On the left is a portion of a type II cell with lamellar bodies (II). Electron micrograph, x 4000

Fig.130 (Below). Apical portion of a type II alveolar cell from rat lung, with microvilli and characteristic surfactantcontaining secretory organelles (lamellar bodies) (arrows). Electron micrograph, x 30000

reason, the fluid will tend to stay within the alveolar wall at first because of the tight epithelial junctions. The fluid can accumulate in the interstitial space containing connective tissue fibers. However, the capillary remains associated with the alveolar epithelium owing to their shared basal lamina, and considerable edema can accumulate without widening of the diffusion barrier, so that gas exchange is maintained until alveolar flooding occurs. The cells of the interstitium are mainly connective tissue cells which differ morphologically from ordinary fibroblasts in several respects. They have a very irregular outline with complex projections of cytoplasm containing discrete bundles of contractile filaments. The cells contact one another through nexus junctions, so that the contraction of several cells is probably functionally coordinated (Bartels 1979). It has been proposed that these cells may have a function in the fine matching of ventilation to perfusion (Kapanci et al. 1974). Alveolar epithelium consists of two types of cells. Roughly 98% of the alveolar surface is covered by squamous (type I) epithelial cells from which thin sheets of cytoplasm extend to cover large areas of surface of one or even several alveoli (Haies et al. 1981; Fig. 129). The other (type II) class of epithelial cell is more numerous but smaller, occupying only 2% of the alveolar surface. Type II epithelial cells can be recognized in the electron microscope by their characteristic lamellar, phospholipid-rich secretory granules, which contain the surface-active alveolar lining material (Askin and Kuhn 1971 ; Fig. 130). In paraffin sections the granules are extracted, and the cells appear vacuolated. In the nonnallung, few cells are found free in the alveolar spaces, and of those which are present, the great majority (more than 90%) are macrophages (Hocking and Golde 1979). Most of the lymphocytes are thymus-derived and present in the same relative proportions as in peripheral blood. Macrophages are continuously removed along the airways toward the nares and must be replaced. The majority probably come from circulating monocytes (van Oud Alblas et al. 1981), but some come from local sources in the lung interstitium (Bowden and Adamson 1980) and perhaps from proliferative pools in the airspaces (Lin et al. 1975). Macrophages produce a variety of secretory products, which are probably important in orchestrating the inflammatory response and repair of injury (Unanue 1976).


References

Mzelius BA (1981) "Immotile-cilia" syndrome and ciliary abnormalities induced by infection and injury. Am Rev Respir Dis 124: 107-109

Askin FB, Kihn C (1971) The cellular origin of pulmonary surfactant. Lab Invest 25: 260-268

Bartels H (1979) Freeze-fracture demonstration of communicating junctions between interstitial cells of the pulmonary interalveolar septa. Am J Anat 155: 125-129

Bensch KG, Gordon GB, Miller LR (1965) Studies on the bronchial counterpart of the Kultschitzky (argentaffin) cell and innervation of bronchial glands. J Ultrastruct Res 12: 668-686

Bowden DH, Adamson IYR (1980) Role of monocytes and interstitial cells in the generation of alveolar macrophages. I. Kinetic studies of normal mice. Lab Invest 42: 511-517

Bowes D, Corrin B (1977) Ultrastructural immunocytochemical localization of lysozyme in human bronchial glands. Thorax 32: 163-170

Boyd MR, Statham CN, Longo NS (1980) The pulmonary Clara cells as a target for toxic chemicals requiring metabolic activation. Studies with carbon tetrachloride. J Pharmacol Exp Ther 212: 109-114

Corkey CW, Levison H, Turner JA (1981) The immotile cilia syndrome. A longitudinal survey. Am Rev Respir Dis 124: 544-548

Cutz E, Chan W, Track NS (1981) Bombesin, calcitonin and leu-enkephalin immunoreactivity in endocrine cells of human lung. Experientia 37: 765-767

Ebert RV, Kronenberg RS, Terracio MJ (1976) Study of the surface secretion of the bronchiole using radioautography. Am Rev Respir Dis 114: 567-573

Erisman MD, Linnoila RI, Hernandez 0, DiAugustine RP, Lazarus LH (1982) Human lung small-cell carcinoma contains bombesin. Proc Nat! Acad Sci USA 79: 2379-2383

Gibbons IR (1981) Cilia and flagella of eukaryotes. J Cell BioI91:107s-124s

Haies DM, Gil J, Weibel ER (1981) Morphometric study of rat lung cells. I. Numerical and dimensional characteristics of parenchymal cell population. Am Rev Respir Dis 123: 533-541

Hijiya K (1978) Electron microscope study of the alveolar brush cell. J Electron Microsc (Tokyo) 27: 223-227

Hocking WG, Golde DW (1979) The pulmonary-alveolar macrophage (Parts I and II). N Engl J Med 301: 580-587,639-645

Horsfield K (1974) The relation between structure and function in the airways of the lung. Br J Dis Chest 68: 145-160

Inoue S, Michel RP, Hogg JC (1976) Zonulae occ1udentes in alveolar epithelium and capillary endothelium of dog lungs studied with the freeze-fracture technique. J UItrastruct Res 56: 215-225

Jeffery PK, Reid L (1975) New observations of rat airway epithelium: a quantitative and electron microscopic study. J Anat 120: 295-320

Kapanci Y, Assimacopoulos A, Ide C, Zwahlen A, Gabbiani G (1974) "Contractile interstitial cells" in pulmonary alveolar septa: a possible regulator of ventilation/ perfusion ratio? Ultrastructural immunofluorescence and in vitro studies. J Cell Bioi 60: 375-392

98 Charles Kuhn III

Kuhn C III, Callaway LA, Askin FB (1974) The formation of granules in the bronchiolar Clara cells of the rat. I. Electron microscopy. J Ultrastruct Res 49 : 387-400

Lauweryns JM, Cokelaere M, Deleersnyder M, Liebens M (1977) Intrapulmonary neuro-epithelial bodies in newborn rabbits. Influence of hypoxia, hyperoxia, hypercapnea, nicotine, reserpine, L-dopa and 5-HTP. Cell Tissue Res 182: 425-440

Lauweryns JM, Cokelaere M, Lerut T, Theunynck P (1978) Cross-circulation studies on the influence of hypoxia and hypoxaemia on neuro-epithelial bodies in young rabbits. Cell Tissue Res 193: 373-386

Lin H-S, Kuhn C, Kuo T-T (1975) Clonal growth of hamster free alveolar cells in soft agar. J Exp Med 142: 877-886

Lindemann CB, Gibbons IR (1975) Adenosine triphosphate-induced motility and sliding of filaments in mammalian sperm extracted with triton X-l00. J Cell BioI 65 : 147-162

Luchtel DL (1978) The mucous layer of the trachea and major bronchi in the rat. Scan Electron Microsc 2: 1089-1099

McDermott MR, Befus AD, Bienenstock J (1982) The structural basis for immunity in the respiratory tract. Int Rev Exp Pathol23: 47-112

Mooren HWD, Meyer CJLM, Kramps JA, Franken C, Dijkman JH (1982) Ultrastructural localization of the low molecular weight protease inhibitor in human bronchial glands. J Histochem Cytochem 30: 1130-1134

Nadel JA, Davis B, Phipps RJ (1979) Control of mucus secretion and ion transport in airways. Annu Rev Physiol 41:369-381

Serabjit-Singh CJ, Wolf CR, Philpot RM, Plopper CG (1980) Cytochrome P-450: localization in rabbit lung. Science 207: 1469-1470

Spicer SS, Chakrin LW, Wardell JR Jr, Kendrick W (1971) Histochemistry of mucosubstances in the canine and human respiratory tract. Lab Invest 25: 483-490

Unanue ER (1976) Secretory function of mononuclear phagocytes. A review. Am J Pathol83: 396-417

van As A (1977) Pulmonary airway clearance mechanisms: a reappraisal. Am Rev Respir Dis 115: 721-726

van Oud Alblas AB, van der Linden-Schrever B, van Furth R (1981) Origin and kinetics of pulmonary macrophages during an inflammatory reaction induced by intravenous administration of heat-killed bacillus CalmetteGuerin. J Exp Med 154: 235-252

Warner FD (1978) Cation-induced attachment of ciliary dynein cross-bridges. J Cell BioI 77: R 19-R26

Weibel ER (1973) Morphological basis of alveolar-capillary gas exchange. Physiol Rev 53: 419-495

NEOPLASMS

Bronchiolar/Alveolar Adenoma, Lung, Rat

Gary A. Boorman

Synonyms. Pulmonary adenoma: alveologenic adenoma.

Gross Appearance

Spontaneous bronchiolar/alveolar adenoma is usually seen as a solitary spherical gray to white smooth nodule on the pleural or cut surface of the lung. Usually the lesion is 1-5 mm in diameter, sharply demarcated from the surrounding lung parenchyma, and often elevated slightly above the pleural surface.


A bronchiolar/alveolar adenoma appears as a focal solid area of increased cellularity that obliterates the underlying alveolar architecture (Fig. 131). The lesion is sharply demarcated from the surrounding tissue, with compression and collapse of adjacent alveoli (Fig. 133). It contains scant connective tissue stroma. Blood vessels are not a prominent feature and relatively few inflammatory cells are seen. In contrast to bronchiolar/ alveolar hyperplasia, in which the cells appear to grow along existing alveolar septa, cells of bronchiolar/alveolar adenoma form solid, glandular or papillary patterns with obliteration of underlying alveolar structures. In the glandular pattern the cells are cuboidal to tall columnar, enclose central lumina (Fig. 132), and have moderate cellular atypia. Mitotic figures are commonly found. The papillary pattern appears similar, with the cells covering a fibrovascular core and forming papillary projections. In some tumors or areas of tumors the cells grow in a solid pattern (Fig. 134) and do not form linear rows. The cells are fairly uniform, with a moderate amount of cytoplasm and poorly defined boundaries. As opposed to the glandular pattern, in which tumor cell nuclei are often oblong, cells in solid areas have round

nuclei of variable size with mild atypia and some mitotic figures. In hematoxylin and eosin sections the cells are usually more basophilic. Important features for distinguishing bronchiolar/alveolar adenoma are distinct borders, compression of adjacent tissue, and obliteration of the underlying architecture.

Ultrastructure

Chemically induced bronchiolar/alveolar adenomas in F 344 rats are characterized by the presence of osmiophilic lamellated organelles, suggesting that the cells are of type II pneumocyte origin (Reznik-Schuller and Reznik 1982). Cells with ultrastructural features of Clara cells, ciliated cells, mucous cells, or APUD-type cells were not found. Ultrastructural studies of the rare spontaneous bronchiolar/alveolar adenomas have not appeared in the literature.


Bronchiolar/alveolar adenoma must be differentiated from bronchiolar/alveolar hyperplasia and bronchiolar/alveolar carcinoma. The adenoma appears morphologically to be a stage in progression from hyperplasia to carcinoma. Thus some confusion exists in distinguishing the benign stage. One might argue that since this process appears to represent a spectrum of lesions progressing to carcinoma, all lesions, even the earliest ones, might logically be called carcinomas. However, it has not been demonstrated that all lesions progress to carcinoma, and some may even disappear when the stimulus is removed. Therefore, it seems prudent to separate lesions into hyperplasia. adenomas, and carcinomas using standard morphological criteria that have been shown to be relevant for other species and organ systems until we have additional information regarding the bio-

100 Gary A. Boorman

Fig.131 (Upper left). Bronchiolar/alveolar adenoma, lung, rat. A well-circumscribed lesion with a glandular pattern. Hand E, x 70

Fig. 132 (Lower left). Bronchiolar/alveolar adenoma with a glandular pattern. The nuclei form serpentine rows and the cells tend to surround lumina. Hand E, x 300

Fig. 133 (Upper right). Bronchiolar/alveolar adenoma that is sharply demarcated from surrounding parenchyma. There is collapse and compression of the adjacent alveoli. Hand E, x 100

Fig. 134 (Lower right). Bronchiolar/alveolar adenoma with a solid pattern. The cells are round and have moderate cytoplasm, poorly defined cell boundaries, and few mitotic figures. The underlying alveolar architecture is not discernible. Hand E, x 240

logic behavior of these lesions. Certainly, a chemical that induces carcinomas with obvious malignant characteristics seems to pose a greater risk to rats than a chemical that after 2 years' exposure only induces lesions that might be called adenomas. Thus separation of these lesions into classical categories may allow a more reasoned judgment of potential hazard of the chemical and certainly will be useful retrospectively as more becomes known about the biologic behavior of these lesions. Bronchiolar/alveolar adenoma can be distinguished from hyperplasia by its sharp demarcation from surrounding parenchyma, loss of underlying alveolar septal architecture and, in some cases, greater cellular atypia. Adenomas tend to undergo a regular pattern of growth, do not stimulate a scirrhous reaction, and do not have the obvious malignant characteristics such as invasion or distant metastases found in carcinomas.

Biologic Features

Bronchiolar/alveolar adenomas are uncommon and were found in only 18 of 2379 (0.8%) female F344 rats and 35 of 2320 (1.5%) male F344 rats in recent 2-year NTP/NCI studies (Haseman et al. 1984). Four adenomas were found in 365 male and one adenoma in 365 female Sprague-Dawley rats in studies terminated at about 2 years (Stula 1975). In the NCI/NTP 2-year toxicology studies, several chemicals were suggested to cause pulmonary tumors in rats (Table 5). In this extensive testing program rats were exposed by gavage, inhalation, dosed feed, dosed water, or skin painting to approximately 300 chemicals, of which 150 were found to be carcinogenic in one or more sex and species. It is interesting to note (Table 5) that in only four cases were the results judged to be positive on the basis of the lung as a target tissue.

Bronchiolarl Alveolar Adenoma, Lung, Rat 101

Table 5. Chemically induced lung tumors in F 344 rats

Chemical Sex Dose Adeno- Carcino-group masb masb

5-Nitroacenaph- M Control 0/96 1/96 (1) thene Low 2/38 (5) 5/38 (13)

High 3/45 (7) 0/45

5-Nitroacenaph- F Control 1/99 (1) 0/99 thene Low 4/46 (9) 4/46 (9)

High 2/31 (6) 1/31 (3)

2,4,5-Trimethyl- F Control 0120 0129 aniline Low 1/43 (2) 2/43 (5)

High 9/50 (18) 2/50 (4)

1,2-Dibromo- F Control 0/50 0/50 ethane" Low 0/48 0/48

High 1/47 (2) 4/47 (9)

" 1,2-Dibromoethane was an inhalation study, the rest of the chemicals were given in the feed. Number of rats with tumors/rats with lung examined

b Number of rats with tumor/rats with lung examined (0/0)

Another important factor would seem to be that only recently have chemicals been given by inhalation. Spontaneous bronchiolar/alveolar adenomas are very uncommon and little is known about their biologic behavior.

References

Haseman JK, Huff JE, Boorman GA (1984) Use of his torical control data in carcinogenicity studies in rodents. Toxicol Pathol (in press)

Reznik-Schuller HM, Reznik G (1982) Morphology of spontaneous and induced tumors in the bronchioloalveolar region of F344 rats. Anticancer Res 2: 53-57

Stula EF (1975) Naturally occurring pulmonary tumors of epithelial origin in Charles-River CD rats. Bull Soc Pharm Environ Pathol3: 3-11

102 Shirley L. Kauffman and Tamiko Sato

Alveolar Type II Cell Adenoma, Lung, Mouse

Shirley L. Kauffman and Tamiko Sato

Synonyms. Type II cell adenoma; pulmonary adenoma; bronchiolo-alveolar adenoma; alveologenic adenoma.

Gross Appearance

Alveolar adenomas are pearly white, well demarcated spherical tumors, visible beneath the pleural surface or protruding from it. The majority measure 2-4 mm in diameter but some reach 1.0 cm. They are typically firm to rubbery, solid, and homogeneous.


Type II adenomas consist of a homogeneous population of uniform-sized cuboidal cells resembling normal type II respiratory epithelium (Figs.135 and 136). They may be situated in any part of the lung parenchyma but are most frequent in the periphery, often near the pleural surface. Individual cells are 121lm with central spherical, hyperchromatic nuclei measuring 5-71lm in diameter. The cytoplasm is eosinophilic in formalin-fixed, paraffin-embedded tissues stained with hematoxylin and eosin, and the char-

acteristic lamellar bodies are represented by fine vacuoles (Fig. 137). The latter stain black in glutaraldehyde-fixed postosmicated tissues embedded in plastic (Fig. 138). The histological pattern is generally trabecular with anastomosing cords forming solid nests; glands or tubules are uncommon except in old tumors. Cell cords are separated on one side by basal alveolar capillaries and sparse reticulin fibers, while the opposite luminal border faces a residual airspace. These unencapsulated tumors have irregular borders created by the extension of neoplastic cells along adjacent alveolar walls. Macrophages containing ingested lipid and inclusions are frequent along the periphery.

Ultrastructure

Type II adenoma cells resemble normal alveolar cells in that they contain characteristic lamellar bodies, large mitochondria (Fig. 139), and prominent Golgi zones (Fig. 140). Tubular nuclear inclusions have been described in both murine and human tumors (Flaks and Flaks 1970; Torikata and Ishiwata 1977): Both type A and type C particles have been demonstrated in carcinogen-induced

Fig. 135. Type II adenoma, lung, mouse. Note discrete cords of type II cells lining alveolar walls. Hand E, x 60

Fig. 136. Type II adenoma with homogeneous cell popula-[> tion, trabecular pattern, and irregular but well-demarcated border. Hand E, x 60

'V Fig.137 (Left) Higher magnification of tumor in Fig.136. Note the large spherical nuclei and abundant eosinophilic, vacuolated cytoplasm typical of type II adenoma cells. H and E, x 500

Fig. 138 (Right). Type II adenoma, lung, mouse. Characteristic lamellar bodies seen at the light microscopic level in tissue fixed in glutaraldehyde and postosmicated, x 500

Alveolar Type II Cell Adenoma, Lung, Mouse 103


Fig.139 (Left). Cells of type II adenoma, lung, mouse. Note large mitochondria and lamellar bodies with parallel membrane stacks. Microvilli line the central portion of the luminal surface; the remainder is covered by flattened surface epithelium. Glutaraldehyde, postosmicated, TEM, x 12000 (reduced by 15%)

type II adenomas (Bucciarelli and Ribacchi 1972) and in carcinomas arising from them (Kimura et al. 1972). Terminal bars are present and cell junctions are relatively straight. The luminal surfaces bear central microvilli, which are covered laterally by cytoplasmic flaps of adjacent type I cells, as in normal lung (Brooks 1968). By scanning electron microscopy (Sato and Kauffman 1980) the tumors are seen to consist of nodular masses, generally spherical, with relatively smooth outer surfaces (Fig. 141). The smooth lining is interrupted by protrusions of the microvillus cap of type II cells through the surrounding type I cytoplasm (Fig. 142).


Type II adenomas should be distinguished from hyperplasia due to toxic injury or viral infection,

Fig. 140 (Right). Apex of a type II adenoma cell with prominent Golgi zone, smooth endoplasmic reticulum, and free ribosomes. TEM, x 12000 (reduced by 15%)

adenomatosis (Hom et al. 1952), and Clara cell adenomas. Distinguishing features of type II adenomas are trabecular pattern, homogeneous cell population, uniform round nuclei and, ultrastructurally, their characteristic lamellar bodies, large mitochondria, and straight plasma membranes. Surfactant-specific apoprotein has been detected in both the nucleus and cytoplasm of human type II adenoma cells (Singh et al. 1981).

Biologic Features

Descriptions of the natural history of mouse lung adenomas frequently include both type II and Clara cell adenomas, as these have only recently been distinguished. Experimentally induced tumors are recognizable days to weeks after carcinogen exposure, and may be preceded by alveolar epithelial hyperplasia (Shimkin 1955; Kauffman 1976). In the early stages, tumors consist of dis-

Fig.141 (Left). Type II cell adenoma, lung, mouse. Smooth-surfaced lobules of type II cells seen protruding from alveoli and alveolar ducts. SEM, x. 450 (reduced by 15%)

crete cords of cuboidal cells lining alveoli (Grady and Stewart 1940) (Fig. 135). Characteristic solid trabecular tumors form as the adjacent affected alveoli collapse. Initial growth is rapid, and volume-doubling time of tumors 1-2 months of age has been estimated as 4.4 days (Shimkin and Polissar 1955). With aging, growth rate progressively decreases, due in part to a decrease in the growth fraction (Dyson and Heppleston 1976). Malignant tumors have been described arising both spontaneously and after carcinogen exposure. These are presumed to arise in preexisting adenomas (see bronchiolar adenomas). Studies of Amaral-Mendes (1969) indicated that approximately 19% of spontaneous alveolar tumors in mice 2 years and older were malignant. Metastases are rare (1%-4% of all lung tumors); most frequent sites are local lymph nodes, heart, and diaphragm (Stewart et al. 1979; Turosov et al. 1974). Established cell lines derived from type II tumors have maintained their differentiated ultrastructural features and secretion of pulmonary surfactant (Stoner et al. 1975).


Fig. 142 (Right). Type II cell adenoma, mouse. A single tumor lobule with discrete protrusions of microvillus caps through the ruffled to flattened epithelium of type I cells. SEM, x 4500 (reduced by 15%)

Etiology and Frequency

Adenomas have been induced by a variety of carcinogens: chemical and physical agents, food additives, and environmental dusts and fumes (Shimkin 1955; Shimkin and Stoner 1975). Data on the frequency of spontaneous lung adenomas (both alveolar and Clara cell) in several mouse strains is shown in Table 6. For all lung adenomas genetic constitution is the main determinant, and frequency ranges from 70% in the most susceptible A strain to 1 % in resistant C 57 BL. Susceptibility of the same strains to carcinogen-induced adenomas parallels natural frequency within the same strain and appears to be regulated by the major histocompatability complex, H2 (Demant and Cleton 1980). The difference in frequency of gross adenomas induced by urethane in sensitive versus relatively resistant strains may. be due to differences in a single gene (Malkinson and Beer 1983). Evaluation of the histologic types of adenomas induced in C 57 strains has demonstrated an association of particular H2 haplotypes with


Table 6. Frequency of pulmonary tumors in eight strains of mice (modified from Shimkin 1955)

Strain

A Swiss BALB c(C) I Y C3H dba CS7 leaden (L or M) CS7 black

Pulmonary tumors per 100 animals 12-18 months old

70-90 40-50 15-25 10-20 10-20 5-15 5 1 1

the development of alveolar type II adenomas (Oomen et al. 1983).


In man, neoplasms arising from type II alveolar cells are classed as a subgroup of pulmonary adenocarcinomas, termed "bronchioloalveolar carcinoma." These are distinguished from the more common terminal bronchiolar carcinomas by their histology, typical osmiophilic lamellar inclusions (Bonikos et al. 1977), and surfactant-specific apoprotein (Singh et al. 1981). Familial cases of alveolar cell carcinoma, with and without coexistent interstitial pulmonary fibrosis, have been described (Beaumont et al. 1981; Joishy et al. 1977). Although the peripheral bronchioloalveolar carcinomas in man are frequently associated with scarred parenchyma and have been termed "scar cancers," no such association has been shown in spontaneous or induced mouse tumors. Adenomas resembling those of mice occur in a variety of birds and animals (Stewart 1966), and the similarity of jaagsiekte, sheep pulmonary carcinoma, to both type II and Clara cell carcinoma of man has been noted (see Clara cell adenoma).

References

Amaral-Mendes JJ (1969) Histopathology of primary lung tumours in the mouse. J Pathol97: 415-427

Beaumont F, Jansen HM, Elema JD, ten Kate LP, Sluiter HJ (1981) Simultaneous occurrence of pulmonary interstitial fibrosis and alveolar cell carcinoma in one family. Thorax 36: 252-258

Bonikos DS, Hendrickson M, Bensch KG (1977) Pulmonary alveolar cell carcinoma. Fine structural and in vitro study of a case and critical review of this entity. Am J Surg Pathol1: 93-108

Brooks RE (1968) Pulmonary adenoma of strain A mice: an electron microscopic study. JNCI 41: 719-742

Bucciarelli E, Ribacchi R (1972) C-type particles in primary and transplanted lung tumors induced in BALBI c mice by hydrazine sulfate: electron microscopic and immunodiffusion studies. JNCI 49: 673-684

Demant P, Cleton FJ (1980) Histocompatibility genes and neoplasia. In: Cleton FJ, Simons JW (eds) Genetic origins of tumor cells. Nijhoff, The Hague, pp 109-125

Dyson P, Heppleston AG (1976) Cell kinetics of urethaneinduced murine pulmonary adenomata. II. The growth fraction and cell loss factor. Br J Cancer 33: 105-111

Flaks B, Flaks A (1970) Fine structure of nuclear inclu-sions in murine pulmonary tumor cells. Cancer Res 30: 1437-1443

Grady HG, Stewart HL (1940) Histogenesis of induced pulmonary tumors in strain A mice. Am J Pathol 16: 417-432 + 3 plates

Horn HA, Congdon CC, Eschenbrenner AB, Andervont HB, Stewart HL (1952) Pulmonary adenomatosis in mice. JNCI 12: 1297-1315

Joishy SK, Cooper RA, Rowley PT (1977) Alveolar cell carcinoma in identical twins. Similarity in time of onset, histochemistry, and site of metastasis. Ann Intern Med 87:447-450

Kauffman SL (1976) Autoradiographic study of type IIcell hyperplasia in lungs of mice chronically exposed to urethane. Cell Tissue Kinet 9: 489-497

Kimura I, Miyake T, Ishimoto A, Ito Y (1972) Intracisternal A-type and C-type particles observed in pulmonary tumors in mice. Gan 63: 563-573

Malkinson AM, Beer DS (1983) Major effect on susceptibility to urethan-induced pulmonary adenoma by a single gene in BALB/cBY mice. JNCI 70: 931-936

Oomen LCJM, Demant P, Hart AAM, Emmelot P (1983) Multiple genes in the H-2 complex affect differently the number and growth rate of transplacentally induced lung tumours in mice. Int J Cancer 31: 447 -454

Sato T, Kauffman SL (1980) A scanning electron microscopic study of the type 2 and Clara cell adenoma of the mouse lung. Lab Invest 43: 28-36

Shimkin MB (1955) Pulmonary tumors in experimental animals. Adv Cancer Res 3: 223-267

Shimkin MB, Polissar MJ (1955) Some quantitative observations on the induction and growth of primary pulmonary tumors in strain A mice receiving urethan. JNCI 16:75-97

Shimkin MH, Stoner GD (1975) Lung tumors in mice: application to carcinogenesis bioassay. Adv Cancer Res 21: 1-58

Singh G, Katyal SL, Torikata C (1981) Carcinoma of type 2 pneumocytes. Immunodiagnosis of a subtype of "bronchioloalveolar carcinomas." Am J Pathol102: 195-208

Stewart" HL (1966) Comparison of histologic lung cancer types in captive wild mammals and birds and laboratory and domestic animals In: Severi L (ed) Lung tumours in animals. Div of Cancer Res, Perugia, pp 25-58

Stewart HL, Dunn TB, Snell KC, Deringer MK (1979) Tumours of the respiratory tract. In: Turusov VS (ed) Pathology of tumours in laboratory animals, vol II, Tumours of the mouse. IARC Sci Pub123: 251-287

Bronchiolar Adenoma, Lung, Mouse

Shirley L. Kauffman and Tamiko Sato

Synonyms. Clara cell adenoma; papillary adenoma; bronchioloalveolar adenoma.

Gross Appearance:

These adenomas are pearly white, spherical, subpleural tumors 2-4 mm in diameter, indistinguishable grossly from type II adenomas. Larger tumors, 0.5-1.0 cm in diameter, are frequently tan. Multiple mulberry-shaped nodules may protrude from the cut surfaces. Adenomas arise in the pleural, subpleural, and deeper parenchyma of the lung, and are equally represented in different lobes of the lung.


Neoplastic Clara cells are cuboidal to columnar, resembling the nonciliated terminal bronchiolar epithelium of normal mouse lung. The nuclei vary in size and in shape from spherical to elongated, and frequently exhibit deep nuclear folds and invaginations. Whereas the cytoplasm appears pale in paraffin-embedded, formalin-fixed tissues stained with hematoxylin and eosin, abundant small vacuoles and osmiophilic particles are demonstrated in tumors fixed by glutaraldehyde and postosmicated (Fig. 143). Clara cell adenomas grow in either a tubular or papillary pattern. In the former, cuboidal or columnar cells are enclosed in cylindrical tubes separated by connective tissue septa (Fig. 143). Macrophages, cell secretion, and debris frequently fill the tubular or


Stoner GD, Kikkawa Y, Kniazeff AJ, Miyai K, Wagner RM (1975) Clonal isolation of epithelial cells from mouse lung adenoma. Cancer Res 35: 2177-2185

Torikata C, Ishiwata K (1977) Intranuclear tubular structures observed in the cells of an alveolar cell carcinoma of the lung. Cancer 40: 1194-1201

Turusov VS, Breslow NE, Tomatis L (1974) Frequency and organ distribution of lung tumor metastases in CF-l mice. JNCI 52: 225-232

Fig. 143. Tubular Clara cell adenoma. Columnar cells with irregular, elongated nuclei line well-formed tubules. Macrophages, cell debris, and secretions are present in the lumen. Numerous small cytoplasmic vacuoles and osmiophilic particles are seen. Glutaraldehyde, postosmicated, Epon-embedded, x 500


Fig. 144. Tubular Clara cell adenoma with several papillary stalks. Secretory activity of the lining cells is evident. Hand E, x 60

acinar spaces. Large tubular tumors may have papillary areas (Fig. 144). Typical mature papillary tumors consist of elongated vascular stalks lined by cuboidal to tall columnar cells with variable amounts of sequestrated secretions (Fig. 145). Clara cell adenomas have no true capsule but their margins are usually clearly outlined by a rim of compressed lung tissue.

Ultrastructure

Striking ultrastructural features of Clara cell adenomas (Kauffman et al. 1979) include nuclear infoldings and complex interdigitations with adjacent cells (Figs.146 and 147). Mitochondria are small in comparison to those of type II adenoma cells, and while these may have ill-defined cristae with dark matrices similar to Clara cells of normal mouse, the matrices are often pale and the cristae prominent (Fig. 146). Endoplasmic reticulum is abundant, glycogen may be present, and secretion granules measuring 300-500 nm and membraneenclosed crystals are characteristic (Fig. 147). My-

Fig. 145. A typical mature papillary adenoma in which vascular stalks, containing dense collagen, are lined by cuboidal to columnar epithelium. Hand E, x 250

elin figures resembling those in injured Clara cells have been described in neoplastic Clara cells (Kennedy et al. 1977). By scanning electron microscopy (Sato and Kauffman 1980) Clara cell tumor surfaces present lobules consisting of closely apposed epithelial cells separated by slightly depressed furrows creating grape-like clusters (Fig.148). Microvilli cover the entire cell surface except for a central apical zone, which is smooth to slightly ruffled (Fig. 149).


Clara cell adenoma should be distinguished from type II alveolar adenoma and adenomatosis, the latter a benign condition in which mucin-containing columnar epithelial cells line alveolar septa (Hom et al. 1952). Mouse Clara cells are mucin negative. Differential features suggesting Clara cell origin include papillary or tubular pattern, heterogeneity of nuclear size and shape, and characteristic nuclear folds. By electron microscopy, typ-

Fig. 146. Electron micrograph of columnar epithelial cells of a papillary adenoma with microvilli along the luminal surfaces, small dense bodies, mitochondria with pale matrices, and abundant endoplasmic reticulum. x 6000

ical features are 300-500 nm secretory granules, crystals, glycogen granules, nuclear invaginations, and complex interdigitating plasma membranes.

Biologic Features

As noted under alveolar adenoma, the natural histories of the two histologic types of adenomas have been treated together in the past. More information is now becoming available regarding the pathogenesis of Clara cell tumors. Clara cell adenomas appear to arise either in the peripheral air sacs or form hyperplastic foci in bronchioles and alveolar ducts (Shami et al. 1982). Kennedy et al. (1977) described bronchiolization of alveoli followed by nodules of hyperplastic Clara cells as precursors of the carcinomas induced in hamsters by polonium-210. Bronchiolar adenomas seldom or never arise within bronchi, although large tumors do protrude into them. With age, merging of adjacent adenomas may convert an entire lung lobe into a multilobulated tumor mass.

Bronchiolar Adenoma, Lung, Mouse 109

Fig. 147. Tubular Clara cell adenoma with irregular infoldings of the nuclear membranes, complex interdigitations of plasma membranes, dense bodies, and membrane-enclosed crystals. x 12000

The latent period is variable, depending upon strain, age, dose, type of carcinogen and route of exposure. Following transplacental carcinogen exposure, tubular clara cell tumors have been recognized in Swiss mice at 10 days of age and papillary tumors after 22 days. Studies of the subsequent progression of these tumors indicated that the papillary tumors arose from an early tubular form (Kauffman 1981). In nude mice, latent periods of up to 9 months have been found for papillary tumors (Anderson 1978). The delayed appearance of papillary compared to alveolar tumors has been noted by several investigators (Grady and Stewart 1940; Kimura 1971). The latter investigator hypothesized a progression from benign to malignant tumors in which alveolar adenomas became papillary and then carcinomatous. Amaral-Mendes, on the other hand, classified adenomas as either of alveolar or bronchiolar origin and found that the latter gave rise to tumors of greater malignancy (Amaral-Mendes 1969). Metastases have been described in 1 %-4% of both


Fig. 148. Scanning electron microscopic view of a papillary adenoma in which large grape-like clusters of Clara cells protrude into an airspace. x 450

spontaneous and induced tumors (Stewart et al. 1979; Turusov et al. 1974). Malignant cell lines have been derived from ethyl-nitrosourea-induced Clara cell tumors, which grow both as subcutaneous papillary and ascites tumors when transplanted to nude mice (Parsa and Kauffman 1983).

Etiology and Frequency

Available data on the etiology (Shimkin 1955; Shimkin and Stoner 1975) and on the frequency of lung adenomas (see alveolar adenoma, Table 6) have not distinguished these according to cell of origin. However, there have been some studies which deal specifically with induction of Clara cell tumors. Adenomas induced in the tree shrew treated with 2,2' -dihydroxy-di-n-propylnitrosamine (Rao and Reddy 1980) and the peripheral tumors arising in hamsters exposed to 210pO were all of Clara cell origin (Kennedy etal. 1977). In

Fig.149. Surfaces of Clara cell clusters covered by microvilli except for smooth central atypical regions. x 4500

the mouse, Clara cell tumors comprised approximately 50% of the total adenomas induced by transplacental exposure to ethyl nitrosourea (Kauffman 1981).


Clara cell carcinomas were first recognized in man (Montes et al. 1966) and comprise one type of bronchioloalveolar tumors of the peripheral adenocarcinoma group (WHO 1981). These grow along the alveolar septa, are frequently papillary, and are mucin positive. Despite some attempts to establish the origin of human bronchioloalveolar carcinomas from one cell type (either type II or Clara cells), current studies indicate the likelihood that there are two distinct tumors. Clara cell tumors have been distinguished from type II carcinomas ultrastructurally by their characteristic nuclear profiles and cytoplasmic granules (Greenberg et al. 1975), and granules of glycoprotein similar to those of normal Clara cell have been

demonstrated by autoradiography in Clara cell tumors (Dermer 1982). Surfactant-specific apoprotein detected in type II tumors has not been demonstrated in several Clara cell tumors (Singh et al. 1981). The naturally occurring pulmonary carcinoma of sheep (jaagsiekte) has ultrastructural features resembling both Clara cell and type II carcinoma and has been proposed as a model of the disease in man (Nobel and Perk 1978). In addition to sheep, domesticated animals with a relatively high incidence of peripheral adenocarcinoma include dogs, cats, horses, and cattle (Balo 1966). In dogs, three-quarters of all primary lung tumors are said to be bronchiolar carcinomas; these are frequently subpleural, may be multicentric, are acinar or papillary with mucin production, and may metastasize (Nielsen 1966).

References

Amaral-Mendes JJ (1969) Histopathology of primary lung tumours in the mouse. J Pathol97: 415-427

Anderson LM (1978) Two crops of primary lung tumors in BALBI c mice after a single transplacental exposure to urethane. Cancer Lett 5: 55-59

Bala J (1966) Comparison of lung tumours in animals and man. In: Severi L (ed) Lung tumours in animals. Div Cancer Res, Perugia, pp 127-139

Dermer GB (1982) Origin of bronchioloalveolar carcinoma and peripheral bronchial adenocarcinoma. Cancer 49:881-887

Grady HG, Stewart HL (1940) Histogenesis of induced pulmonary tumors in strain A mice. Am J Pathol 16: 417-432 + 3 plates

Greenberg SD, Smith MN, Spjut HJ (1975) Bronchioloalveolar carcinoma - cell of origin. Am J Clin Pathol63: 153-167

Hom HA, Congdon CC, Eschenbrenner AB, Andervont HB, Stewart HL (1952) Pulmonary adenomatosis in mice. JNCI 12: 1297-1315

Kauffman SL (1981) Histogenesis of the papillary Clara cell adenoma. Am J Pathol103: 174-180

Kauffman SL, Alexander L, Sass L (1979) Histologic and ultrastructural features of the Clara cell adenoma of the mouse lung. Lab Invest 40: 708-716

Bronchiolar Adenoma, Lung, Mouse 111

Kennedy AR, McGandy RB, Little JB (1977) Histochemical, light and electron microscopic study of polonium-210 induced peripheral tumors in hamster lungs: evidence implicating the Clara cell as the cell of origin. Eur J Cancer 13: 1325-1340

Kimura I (1971) Progression of pulmonary tumor in mice. I. Histological studies of primary and transplanted pulmonary tumors. Acta Pathol Jpn 21: 13-56

Montes M, Adler RH, Brennan JC (1966) Bronchiolar apocrine tumor. Am Rev Respir Dis 93: 946-950

Nielsen SW (1966) Spontaneous canine pulmonary tumors. In: Severi L (ed) Lung tumours in animals. Div Cancer Res, Perugia, pp 151-164

Nobel TA, Perk K (1978) Bronchiolo-alveolar cell carcinoma. Animal model: pulmonary adenomatosis of sheep, pulmonary carcinoma of sheep (jaagsiekte). Am J Pathol90: 783-786

Pars a I, Kauffman SL (1983) Malignant Clara cell line derived from ethyl-nitrosourea-induced murine lung adenomas. Cancer Lett 18: 311-316

Rao MS, Reddy JK (1980) Carcinogenicity of 2,2'-dihydroxy-di-n-propylnitrosamine in the tree shrew (Tupaia glis): light and electron microscopic features of pulmonary adenomas. JNCI 65: 835-840

Sato T, Kauffman SL (1980) A scanning electron microscopic study of the type 2 and Clara cell adenoma of the mouse lung. Lab Invest 43: 28-36

Shami SG, Thibodeau LA, Kennedy AR, Little JB (1982) Proliferative and morphological changes in the pulmonary epithelium of the Syrian golden hamster during carcinogenesis initiated by 21oPoa-radiation. Cancer Res 42: 1405-1411

Shimkin MB (1955) Pulmonary tumors in experimental animals. Adv Cancer Res 3: 223-267

Shimkin MB, Stoner GD (1975) Lung tumors in mice: application to carcinogenesis bioassay. Adv Cancer Res 21:1-58

Singh, G, Katyal SL, Torikata C (1981) Carcinoma of type 2 pneumocytes. Immunodiagnosis of a subtype of "bronchioloalveolar carcinomas". Am J Pathol 102: 195-208

Stewart HL, Dunn TB, Snell KC, Deringer MK (1979) Tumours of the respiratory tract. In: Turusov VS (ed) Pathology of tumours in laboratory animals, vol II. Tumours of the mouse. IARC Sci Pub123: 251-287

Turusov VS, Breslow NE, Tomatis L (1974) Frequency and organ distribution of lung tumor metastases in CF-1 mice. JNCI 52: 225-232

WHO (1981) Histological typing of lung tumours. Tumori 67: 253-272

112 Gary A. Boorman

Bronchiolar/ Alveolar Carcinoma, Lung, Rat

Gary A. Boorman

Synonyms. Bronchiolar carcinoma; alveolar cell carcinoma; adenocarcinoma; carcinoma.

Gross Appearance

While small tumors may not be visible to the unaided eye, most bronchiolar/alveolar carcinomas are of sufficient size to be easily seen. They appear as firm, pale, white to pink irregular smooth nodules projecting above the pleural surface. In advanced cases an entire lobe may be replaced by tumorous tissue. While in man they are most often found in the upper lobe, a site propensity has not been reported for the rat. When cut most will-have a smooth surface, except for those containing areas of necrosis, which will appear grossly as yellow to pale brown irregular areas of soft caseous material within the tumor.


Spontaneous bronchiolar/alveolar carcinomas are usually solitary tumors that tend to occur in the peripheral regions of the lung (Fig. 150). The tumors often are not well circumscribed and invade pleura, vessels, or adjacent bronchi and bronchioles (Fig. 151). The tumor cells may grow along existing alveolar structures or form solid, papillary, and glandular patterns which obliterate the underlying alveolar architecture. The cells are round (in solid patterns) or columnar (papillary or glandular patterns) (Fig. 152), with moderate amounts of cytoplasm and poorly defined cell boundaries. In less differentiated tumors the cells are more pleomorphic and spindle-shaped. These tumors appear to incite a scirrhous response. In some tumors, areas of squamous metaplasia may be found (Fig. 153).

Ultrastructure

Spontaneous and induced bronchiolar/alveolar tumors in the F344 rat are characterized by the presence of intracytoplasmic bodies with crossbarred parallel lamellae analogous to those found in type II pneumocytes (Reznik-SchUller and Reznik 1982).

Fig.iSO. Bronchiolar/alveolar carcinoma obliterating normal alveolar architecture. Hand E, x 120


Bronchiolar/alveolar carcinoma must be differentiated from bronchiolar/alveolar adenoma, metastatic adenocarcinoma, and squamous cell carcinoma. Bronchiolar/alveolar adenomas tend to be better differentiated and lack obvious malignant characteristics such as distant metastases or local invasion. Size is important, since lesions greater than 1 cm in diameter can usually be shown to have areas suggestive of local invasion. Metastatic carcinomas from the reproductive tract or gastrointestinal tract or of bile duct origin can be mistaken for bronchiolar/alveolar carcinoma. Metastatic carcinomas are often multiple, tend to occur in lymphatics or blood vessels, and have a more discrete margin. Usually, at the edge of a bronchiolar/alveolar carcinoma the tumor cells can be found growing along existing alveolar

Bronchiolar/Alveolar Carcinoma, Lung, Rat 113

Fig.1S1. Cells of bronchiolar/alveolar carcinoma invading wall of large bronchus (L, lumen of bronchus; M, smooth muscle wall of bronchus). Hand E, x 120

Fig.1S2. Cells of bronchiolar/ alveolar carcinoma forming a glandular pattern. The cells surrounding lumina tend to be columnar. Hand E, x 400

Fig.1S3. Bronchiolar/alveolar carcinoma with area of squamous metaplasia (arrowhead). Hand E, x 200

114 Gary A. Boorman

structures, while in metastatic tumors a sharper demarcation exists between tumor cells and normal pulmonary tissue. Abundant mucus production is rare in bronchiolar/alveolar carcinomas, but may be a prominent feature in many adenocarcinomas that can metastasize to the lung. A careful search for a primary tumor in other organs or tissues is a necessary prerequisite before accepting the diagnosis of primary bronchiolar/ alveolar carcinoma. Finally, this tumor must be differentiated from squamous cell carcinoma. In man, carcinomas showing features of both squamous cell carcinoma and adenocarcinoma behave as adenocarcinoma (WHO 1982). In the rat, tumors comprising a mixture of squamous cells and bronchiolar/alveolar cells should be diagnosed as bronchiolar/alveolar carcinoma. The diagnosis of squamous cell carcinoma should be reserved for tumors composed entirely of squamous cells and felt to be of bronchogenic origin. Squamous cell carcinomas meeting these criteria are rare in the rat, but when found should be more central in location than bronchiolar/alveolar carcinomas, with local areas of squamous differentiation.

Biologic Features

Morphologically, bronchiolar/alveolar carcinoma appears to represent a progression of the bronchiolar/alveolar adenoma. The exact biologic behavior has not been established, and whether these lesions are malignant from their inception or undergo malignant changes with time has not been established. The limited ultrastructural studies (Reznik-Schuller et al. 1981; ReznikSchuller and Reznik 1982) suggest that some, if not most, of these tumors arise from type Ilpneumocytes. In man, bronchiolar/alveolar carcinomas are placed in a subgroup of pulmonary adenocarcinomas (WHO 1982) that apparently arise from alveoli or bronchioles. Acinar adenocarcinomas are another subgroup of pulmonary adenocarcinomas and may be of bronchial origin (WHO 1982; Dermer 1982). Almost all rat tumors appear to be of the bronchiolar/alveolar type, although one rat was reported to have a mucinous bronchiolar carcinoma with tall columnar cells and mucus production more analogous to the human acinar adenocarcinoma (Yang and Grice 1965). Bronchiolar/alveolar carcinomas have a low tendency to metastasize; of 21 pulmonary carcinomas found in 3099 male and female F344 rats, only one metastasized to the me-

diastinum (Goodman et al. 1980). Two other cases of pulmonary tumor metastases have been reported in the F344 rat (Reznik 1981). Spontaneous bronchiolar/alveolar tumors are infrequent in most rat strains (Burek and Hollander 1977; Table 7). They are reported to be induced by exposure to nickel carbonyl, beryllium sulfate, and chrysotile asbestos (Kuschner and Laskin 1970).


In man, bronchiolar/alveolar carcinoma is one of the least common of the primary lung cancers, accounting for only 5% ofthetotal cases (Greenberg 1982). While the cell of origin has been controversial, it appears that both Clara cells and type II pneumocytes can give rise to this tumor (Greenberg 1982; Dermer 1982; Lieber et al. 1976; Singh et al. 1981; Schraufnagel et al. 1982). In the mouse bronchiolar/alveolar carcinomas are quite common, depending upon the strain, and arise from both Clara cells and type II pneumocytes (Kauffman 1981). In both the dog and cat approximately three-fourths of the pulmonary tumors are classified as adenocarcinomas, while only 26 of 171 pulmonary tumors in dogs and five of 47 pulmonary tumors in cats were considered to be of alveogenic origin (Moulton et al. 1981). Ultrastructural studies suggest that at least some of the rat tumors are of type II pneumocyte origin (ReznikSchuller and Reznik 1982).

References

Anver MR, Cohen Bl, Lattuada CP, Foster SI (1982) Ageassociated lesions in barrier-reared male Sprague-Dawley rats: a comparison between Hap: (SD) and Crl: COPS(R)CD(R)(SD) stocks. Exp Aging Res 8: 3-24

Boorman GA, Hollander CF (1973) Spontaneous lesions in the female W AG/Rij (Wistar) rat. 1 Gerontol 28: 152-159

Burek ID, Hollander CF (1977) Incidence patterns of spontaneous tumors in BN/Bi rats. INCI 58: 99-105

Coleman GL, Barthold SW, Osbaldiston GW, Foster SF, lonas AM (1977) Pathological changes during aging in barrier-reared Fischer 344 male rats. 1 Gerontol 32: 258-278

Dermer GB (1982) Origin of bronchioloalveolar carcinoma and peripheral bronchial adenocarcinoma. Cancer 49:881-887

Goodman DG, Ward 1M, Squire RA, Paxton MB, Reichardt WD, Chu KC, Linhart MS (1980) Neoplastic and nonneoplastic lesions in aging Osborne-Mendel rats. Toxicol Appl Pharmacol55: 433-447

Greenberg SD (1982) Histology and ultrastructure ofbronchiolo-alveolar carcinoma. In: Shimosato Y, Melamed

Bronchiolar/Alveolar Carcinoma, Lung, Rat 115

Table 7. Naturally occurring bronchiolar/alveolar tumors in rats

Strain Sex No. Age Tumors' Reference

Adenomas Carcinomas

ACIIN M 55 Life span 0 0 Maekawa and Odashima 1975

ACIIN F 209 Life span 0 0 Maekawa and Odashima 1975

F344 M 2305 24mo 35 (1.5) 18 (0.8) Haseman et al. 1984 F344 F 2354 24mo 20 (0.9) 9 (0.4) Haseman et al. 1984 F344 M 96 Life span 0 0 Jacobs and Huseby 1967 F344 F 102 Life span 0 0 Jacobs and Huseby 1967 F344 M 160 Life span 0 0 Sass et al. 1975 F344 F 192 Life span 0 0 Sass et al. 1975 F344 M 144 Life span 0 0 Coleman et al. 1977 Holtzman-SD M/F 268 24mo 0 0 MacKenzie and Gamer 1973 Oregon M/F 673 24mo 0 1 (0.1) MacKenzie and Gamer 1973 Osbome-Mendel M 50 24mo 0 0 Radomski et al. 1965 Osbome-Mendel F 50 24mo 0 1 (2) Radomski et al. 1965

Osbome-Mendel M 975 24mo 4 (0.4) 5 (0.5) Goodman et al. 1980 Osbome-Mendel F 970 24mo 2 (0.2) 3 (OJ) Goodman et al. 1980 Sherman M 60 24mo 0 0 Kociba et al. 1974 Sherman F 60 24mo 0 0 Kociba et al. 1974 Sprague-Dawley M 179 18mo 1 (0.6) 2 (1.1) Prejean et al. 1973

Sprague-Dawley F 181 18mo 0 1 (0.5) Prejean et al. 1973

Sprague-Dawley M 85 24mo 1 (1.2) 1 (1.2) Kociba et al. 1978 Sprague-Dawley F 86 24mo 0 0 Kociba et al. 1978

Sprague-Dawley HAP M 45 18-38mo 1 (2.2) 1 (2.2) Anver et al. 1982 (SD)

Sprague-Dawley Crl: M 84 18-38 mo 0 0 Anver et al. 1982 COPS (SD)

Sprague-Dawley M 655 Life span 2 (0.3)b 2 (O.W Ross and Bras 1965 Wistar M 150 24mo 0 0 Torkelson et al. 1974 Wistar F 139 24mo 0 0 Torkelson et al. 1974

Wistar M 472 30mo 0 1 (0.2) Kroes et al. 1981

Wistar F 457 30mo 0 0 Kroes et al. 1981 Wistar F 290 Life span 0 0 Boorman and Hollander

1973

• Total tumors found (%) b All tumors found in the 210 animals receiving high-protein diet

116 Gary A. Boorman

MR, Nettesheim P (eds) Morphogenesis oflung cancer, voI1.CRC, Boca Raton, pp 121-145

Haseman JK. Huff JE, Boorman GA (1984) Use of his torical control data in carcinogenicity studies in rodents. Toxicol Pathol in press

Jacobs BB, Huseby RA (1967) Neoplasms occurring in aged Fischer rats with special reference to testicular, uterine and thyroid tumors. JNCI 39: 303-309

Kauffman SL (1981) Histogenesis of the papillary Clara cell adenoma. Am J Pathol 103: 174-180

Kociba RJ, McCollister SB, Park C, Torkelson TR, Gehring PJ (1974) 1,4-Dioxane. I. Results of a 2-year ingestion study in rats. Toxicol Appl Pharmacol 30: 275-286

Kociba RJ, Keyes DG, Beyer JE, Carreon RM, Wade CE, Dittenber DA, Kalnins RP, Frauson LE, Park CN, Barnard SD, Hummel RA, Humiston CG (1978) Results of a two-year chronic toxicity and oncogenicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats. Toxicol Appl Pharmacol 46: 279-303

Kroes R, Garbis-Berkvens JM, de Vries T, van Nesselrooy HJ (1981) Histopathological profile of a Wistar rat stock including a survey of the literature. J Gerontol 36: 259-279

Kuschner M, Laskin S (1970) Pulmonary epithelial tumors and tumor-like proliferations in the rat. In: Nettesheim P, Hanna MG Jr, Deatherage JW (eds) Morphology of experimental respiratory carcinogenesis, AEC Symposium Series 21 USAEC, Oak Ridge, pp 202-226

Lieber M, Smith B, Szakal A, Nelson-Rees W, Todaro G (1976) A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Int J Cancer 17: 62-70

Mac Kenzie WF, Gamer FM (1973) Comparison of neoplasms in six sources of rats. JNCI 50: 1243-1257

Maekawa A, Odashima S (1975) Spontaneous tumors in ACI/N rats. JNCI 55: 1437-1445

Moulton JE, von Tscharner C, Schneider R (1981) Classification oflung carcinomas in the dog and cat. Vet Pathol 18: 513-528

Prejean JD, Peckham JC, Casey AE, Griswold DP, Weisburger EK. Weisburger JH (1973) Spontaneous tumors in Sprague-Dawley rats and Swiss mice. Cancer Res 33: 2768-2773

Radomski JL, Deichmann WB, MacDonald WE, Glass EM (1965) Synergism among oral carcinogens. Toxicol AppIPharmacoI7:652-656

Reznik G (1981) Unusual sites of lung tumor metastases in B6C3F1 mice and F344 rats. Anticancer Res 1: 159-162

Reznik-SchUller HM, Reznik G (1982) Morphology of spontaneous and induced tumors in the bronchioloalveolar region of F344 rats. Anticancer Res 2: 53-57

Reznik-SchUller HM, Hague BR Jr, Creasia DA (1981) Ultrastructure of pulmonary tumors induced in rats by Nnitrosomethylurethane. J Toxicol Environ Health 8: 501-506

Ross MH, Bras G (1965) Tumor incidence patterns and nutrition in the rat. J Nutr 87: 245-260

Sass B, Rabstein LS, Madison R, Nims RM, Peters RL, Kelloff GJ (1975) Incidence of spontaneous neoplasms in F344 rats throughout the natural life span. JNCI 54: 1449-1456

Schraufnagel D, Peloquin A, Pare JA, Wang NS (1982) Differentiating bronchioloalveolar carcinoma from adenocarcinoma. Am Rev Respir Dis 125: 74-79

Singh G, Katyal SL, Torikata C (1981) Carcinoma of type II pIieumocytes. Am J Pathol102: 195-208

Torkelson TR, Leong BKJ, Kociba RJ, Richter WA, Gehring PJ (1974) 1,4-Dioxane. II. Results of a 2-year inhalation study in rats. Toxicol Appl Pharmacol 30: 287-298

WHO (1982) Histological typing of lung tumors. Neoplasma 29: 111-123

Yang YH, Grice HC (1965) Mucinous bronchiolar carcinoma of the rat lung: a case report. Can J Comp Med 29: 15-17

Squamous Cell Carcinoma, Lung, Syrian Hamster 117

Squamous Cell Carcinoma, Lung, Syrian Hamster

Parviz M. Pour and Hildegard M. Reznik-Schuller

Synonyms. Squamous cell tumor; epidermoid carcmoma.

Gross Appearance

Tumors of 0.5 mm in diameter can be recognized by careful examination of the cut section of the lungs. They may be situated along the bronchi or be randomly distributed all over the lung parenchyma. There is a tendency of tumors to flow together and in some cases a whole lobe may be involved. Small nodules are usually well demarcated, fleshy, and firm and the appearance of their cut surface depends on tumor type. Pure squamous cell tumors are often grayish white and granular on the cut surface. Larger tumors may be cystic or may undergo central necroses. In mixed squamous-adenocarcinomas a viscous fluid may be wiped from the cut surface.


Histologic demonstration of the lesion can be facilitated by placing individually dissected lobes of the hamster lung in fixative. For histologic processing the fixed lung should be cut along the course of the main bronchi in order to include the bronchi in the sections. As in the upper respiratory tract, malignant squamous cells arise from bronchial or bronchiolar epithelial cells which have undergone metaplasia. These malignant cells, in the form of small or large islands, expand into the surrounding alveoli (Fig. 154), gradually replacing the alveolar epithelium and filling out the alveolar lumen. Small foci of tumor cells can often be found within the pulmonary parenchyma, remote from the bronchi (Fig. 155), a finding which may indicate their primary development from alveolar epithelium. However, the possibility that they represent intraluminal growth from a terminal bronchus or plugging of a bronchus by metastases of laryngeal or tracheal lesions cannot always be excluded. Virusand benzo(a)pyrene-induced tumors have been thought to originate from bronchioli, and others, produced by external radiation, from the alveolarbronchiolar epithelium. Pulmonary squamous cell carcinomas occur in remarkably varied patterns and include well or

moderately differentiated, keratinizing types (Fig. 156); nonkeratinizing, transitional cell types; mixed glandular, adenosquamous cell or mucoepidermoid cell types; and anaplastic types. Some tumors present keratin-filled, occasionally large cysts (distended bronchi ?), from the walls of which tumor cells begin to invade (Fig. 157). Squamous cell differentiation can also be observed within or in the vicinity of adenocarcinomas and anaplastic carcinomas (Fig.158), findings which support the concept that all these tumors originate from common stem cells that differentiate into several cell types, including neuroendocrine cells.

Ultrastructure

Multiple focal hyperplasias in segmental bronchi and bronchioles (Fig. 159) have been identified by electron microscopy as proliferating endocrine (APUD-type) cells [for N-nitrosodiethylamine (DEN) and N-nitrosodibuthylamine (DBN) at weeks 2-3, for N-nitrosomorpholine (NM) at weeks 4-5] (see Table 1 for abbreviations). The characteristic dense-cored granules of these endocrine cells decreased in number under continued nitrosamine treatment and were replaced by bundles of cytoplasmic tonofilaments (Fig. 160). The formation of these bundles is the ultrastructural marker for early squamous metaplasia. Lung tumors contained areas with ultrastructural features of endocrine and squamous cells.


Primary squamous cell carcinomas can be distinguished from secondary tumors in most but not all cases. The presence of tumor cells in blood vessels and subpleural locations favors their metastatic nature, but small intraalveolar lesions are at times extremely difficult to distinguish from aspirated pharyngolaryngotracheal neoplasms. Aspirated tumor cells may evoke inflammatory reactions; however, this can also occasionally be seen around primary tumors (Figs. 155 and 160). A decision can often be reached by determining the general pattern of induced tumors. Granulomas associated with calcification of the

118 Parviz M. Pour and Hildegard M. Reznik-Schuller

Fig. 154 (Above). Lung, hamster. Squamous metaplasia of a terminal bronchus. Note part of the original bronchus {arrow}. metaplastic epithelium of the bronchus, and squamous cell nests beneath it. Hand E, x 195

Fig. 155 (Below). Lung, hamster. Islands of malignant squamous cells within the pulmonary parenchyma with no obvious relation to the bronchus {right}. Note inflammatory reaction around the tumor. Hand E, x 78

Fig. 156 (Above). Lung, hamster. Squamous cell carcinoma with limited tendency to keratinize. Hand E, x 195


Fig. 157 (Below). Lung, hamster. Beginning of alveolar invasion by a well-differentiated and cystic squamous cell carcinoma. Hand E, x 195

120 Parviz M. Pour and Hildegard M. Reznik-Schuller

Fig. 158. Anaplastic carcinoma, lung, hamster, composed oflarge and bizarre cells, one with squamous cell differentiation (arrow). Hand E, x 390

bronchial and bronchiolar basal membranes may simulate pleomorphic tumors (Pour and Birt 1979).

Biologic Features

Squamous cell carcinomas are unknown as a spontaneous entity in Syrian hamsters. Therefore, all our comments refer to tumors experimentally induced by one or more carcinogens.

Pathogenesis. The pathogenesis of bronchial tumors induced in Syrian golden hamsters by subcutaneous treatment with DEN (see Table 1) (given at the rate of 17.8 mg/kg body wt. twice weekly), DBN (351 mg/kg given once weekly), and NM (98 mg/kg once a week) have been studied in serial sacrifice experiments by light and electron microscopy (Reznik-Schuller 1976, 1977a, b, 1983a, b; Reznik-Schuller and Reznik 1979). Each of these three nitrosamines induced the same spectrum of lesions. However, the latency period was different for each compound. Ultrastructural findings parallel observations of

multidirectional differentiation in human lung tumors, in which admixtures of squamous, endocrine, and glandular features may be found (Churg et al. 1980; Gould et al. 1981). Moreover, the results demonstrate that these nitrosamines can selectively induce multiple proliferations of the otherwise sparse endocrine cells in the hamster lung. This model system has been successfully used for the in vitro cultivation of pulmonary endocrine cells (Linnoila 1982; Linnoila et al. 1981), and makes studies of the cells of origin of human oat cell carcinoma (pulmonary endocrine cells) possible.

Incidence. As with other respiratory tract tumors, the incidence of pulmonary squamous cell carcinomas varies in relation to the specificity and potency of the carcinogen for the pulmonary tissue. Among carcinogens tested N-nitrosodi-n-propylamine (DPN) was more effective than its assumed beta-metabolites for inducing this tumor type (Althoff et al. 1977 a; Pour et al. 1973, 1974 a). The most potent carcinogen was N-nitrosovinylethyl amine, (VEN) which induced incidences of 75% squamous cell carcinomas and 17% adeno-

Fig. 159. Segmental bronchus of a hamster after 4 weeks of administration of N -nitrosodiethylamine. In the basal epithelial layer, note proliferated cells with ultrastructural features of neuroendocrine (APUD-type) cells. Uranyl acetate and lead citrate. TEM, x 1700

Fig. 160. Cell of segmental bronchus of a hamster that received N-nitrosomorpholine for 12 weeks. In this neuroendocrine cell, many of the dense-cored granules have been replaced by tonofilament bundles. Uranyl acetate and lead citrate, TEM, x 12000 (reduced by 15%)


122 Parviz M. Pour and Hildegard M. Reznik-SchUller

squamous cell carcinomas (Althoff et al. 1977b). DEN also resulted in induction of a number of pulmonary squamous cell carcinomas (Dontewill and Mohr 1961; Herrold and Dunham 1963; Mohr 1970; Althoff et al. 1971) Other carcinogens, such as N-nitrosobis(2-acetoxypropyl)amine (BAP) (Pour et al. 1976) and N-nitrosohexamethyleneimine (N-6-MI) (Althoff et al. 1973), are relatively weak inducers of these neoplasms and N-nitrosomethyl(2-oxopropyl)amine (MOP) (Pour et al. 1980), N-nitrosobis(2-hydroxypropyl)amine (BHP) (Pour et al. 1975), N-nitroso-2-oxopropyl-n-propylamine (2-0PPN) (Pour et al. 1974a), N-nitroso-2-hydroxypropyl-n-propylamine (2-HPPN) (Pour et al. 1974b), and N-nitrosomethyl-n-propylamine (MPN) (Pour et al. 1974c) were ineffective, although these carcinogens, especially BHP, have induced a high incidence of papillomas and carcinomas in the upper respiratory tract. Alkylnitrosamines with one methyl group in one aliphatic chain in an alphaposition apparently lose their specificity for the lungs, but gain potency for the nasal epithelium (Pour et al. 1980) Among other carcinogens, external radiation (DeVilliers and Gross 1966), 9,10-dimethyl-1,2-benzanthracene (DMBA) Gross et al. 1965), and benzo(a)pyrene (BaP) have been reported as potent inducers of pulmonary squamous cell carcinomas (Herrold and Dunham 1963; Dontenwill and Mohr 1962; Saffiotti 1970; Saffiotti et al. 1964, 1966, 1972a, b; Henry and Kaufman 1973; Henry et al. 1973, 1975; Schreiber et al. 1974; Reznik-Schuller and Mohr 1974, 1975). Viral infections have been reported to be etiologic agents also (Rabson et al. 1960).

Sex. No clear-cut sex differences in terms of tumor incidence or predominant tumor types have been observed, either in our series or in those from other laboratories. Squamous cell carcinomas grow expansively. It is difficult to define invasion in tissues such as lungs, which are composed of intercommunicating sacs without limiting capsules. However, in general these lesions, in contrast to adenomas, are not well circumscribed. We consider any squamous cell tumor of the lungs to be a carcinoma, regardless of size. Despite their local invasive behavior, the metastasis of such tumors must be extremely rare, and was not observed in our material.

References

Althoff J, Cardesa A, Pour P, Mohr U (1973) Carcinogenic effect of n-nitrosohexamethylenimine in Syrian golden hamsters. JNCI 50: 323-329

Althoff J, Grandjean C, Pour P, Bertram B (1977 a) Comparison of the effect of beta-oxidized dipropylnitrosamine metabolites administered at equimolar doses to Syrian hamsters. Z Krebsforsch 90: 141-148

Althoff J, Grandjean C, Russell L, Pour P (1977b) Vinylethylnitrosamine: a potent respiratory carcinogen in Syrian hamsters. JNCI 58: 439-442

Althoff J, Wilson R, Mohr U (1971) Diethylnitrosamineinduced alterations in the tracheobronchial system of Syrian golden hamster. JNCI 46: 1067-1071

Churg A, Johnston WH, Stulbarg M (1980) Small cell squamous and mixed small cell squamous-small cell anaplastic carcinomas of the lung. Am J Surg Pathol 4: 255-263

De Villiers AJ, Gross P (1966) Morphologic changes induced in the lungs of hamsters and rats by external radiation (x-rays). A study in experimental carcinogenesis. Cancer 19: 1399-1410

Dontenwill W, Mohr U (1961) Carcinome des Respirationstraktus nach Behandlung von Goldhamstern mit Diathylnitrosamin. Z Krebsforsch 64: 305-312

Dontenwill W, Mohr U (1962) Vergleichende Untersuchungen an metaplastichen und malignen Epithelveranderungen des Respirationstraktes im Tierexperiment. Z Krebsforsch 65: 168-170

Gould VE, Memoli VA, Dardi LE (1981) Multidifferentiation in human epithelial cancers. J Submicro Cytol 13: 97-103

Gross P, Tolker E, Babyak MA, Kaschak M (1965) Experimental lung cancer in hamsters. Arch Environ Health 11:59-65

Henry MC, Kaufman D (1973) Clearance of benzo(a)pyrene from hamster lungs after administration on coated particles. JNCI 51: 1961-1964

Henry MC, Port CD, Bates RR, Kaufman DG (1973) Respiratory tract tumors in hamsters induced by benzo( aJpyrene. Cancer Res 33: 1585-1592

Henry MC, Port CD, Kaufman DG (1975) Importance of physical properties of benzo(aJpyrene-ferric oxide mixtures in lung tumor induction. Cancer Res 35: 207-217

Herrold KM, Dunham U (1963) Induction of tumors in the Syrian hamster with diethylnitrosamine (N-nitrosodiethylamine). Cancer Res 23: 773-777

Linnoila RI (1982) Effects of diethylnitrosamine on lung neuroendocrine cells. Exp Lung Res 3: 225-236

Linnoila RI, Nettesheim P, DiAugustine RP (1981) Lung endocrine-like cells in hamsters treated with liiethylnitrosamine: alterations in vivo and in cell culture. Proc Nat! Acad Sci USA 78: 5170-5174

Mohr U (1970) Effects of diethylnitrosamine in the respiratory system of Syrian golden hamsters In: Nettesheim P, Hanna MG, Deatherage JW (eds) Morphology of experimental respiratory carcinogenesis, AEC Symposium Series No.21. USAEC, Div Tech Info Ext, Oak Ridge, pp 255-265

Pour P, Birt D (1979) Spontaneous diseases of Syrian golden hamsters - their implications in toxicological re-

search: facts, thoughts and suggestions. Prog Exp Tumor Res 24: 145-156

Pour P, KrUger FW, Cardesa A, Althoff J, Mohr U (1973) Carcinogenic effect of di-n-propylnitrosamine in Syrian golden hamsters. JNCI 51: 1019-1027

Pour P, Althoff J, Cardesa A, KrUger FW, Mohr U (1974 a) Effect of beta-oxidized nitrosamines on Syrian golden hamsters. II. 2-0xopropyl-n-propylnitrosamine. JNCI 52: 1869-1874

Pour P, KrUger FW, Althoff J, Cardesa A, Mohr U (1974b) Effect of beta-oxidized nitrosamines on Syrian golden hamster. I. 2-Hydroxypropyl-n-propylnitrosamine. JNCI 52: 1245-1249

Pour P, KrUger FW, Cardesa A, Althoff J, Mohr U (1974 c) Tumorigenicity of methyl-n-propylnitrosamine in Syrian golden hamsters. JNCI 52: 457-462

Pour P, KrUger FW, Althoff J, Cardesa A, Mohr U (1975) Effect of beta-oxidized nitrosamines on Syrian hamsters. III. 2,2'-Dihydroxy-di-n-propylnitrosamine. JNCI 54:141-146

Pour P, Althoff J, Gingell ~ Kupper ~ KrUger F, Mohr U (1976) n-Nitroso-bis(2-acetoxypropyl)amine as a further pancreatic carcinogen in Syrian golden hamsters. Cancer Res 36: 2877-2884

Pour P, Gingell ~ Langenbach ~ Nagel D, Grandjean C, Lawson T, Salmasi S (1980) Carcinogenicity of N-nitrosomethyl(2-oxopropyl)amine in Syrian hamsters. Cancer Res 40: 3585-3590

Rabson AS, Branigan WJ, Legallais FY (1960) Lung tumors produced by intratracheal inoculation of polyoma virus in Syrian hamsters. JNCI 25: 937-965

Reznik-Schuller H (1976) Proliferation of endocrine (APUD-type) cells during early N-diethylnitrosamineinduced lung carcinogenesis in hamsters. Cancer Lett 1 : 255-258

Reznik-Schuller H (1977 a) Ultrastructural alterations of APUD cells during early N-diethylnitrosamine-induced carcinogenesis. J Pathol 121: 79-82

Reznik-Schuller H (1977 b) Sequential morphologic alterations in the bronchial epithelium of Syrian golden hamster during N-nitrosomorpholine-induced pulmonary tumorigenesis. Am J Pathol 89: 59-66

Reznik-SchUller HM (1983 a) Cancer induced in the respiratory tract of rodents by N-nitroso compounds. In: Reznik-SchUller HM (ed) Comparative respiratory tract carcinogenesis, vol 2, Experimental respiratory tract carcinogenesis. CRC, Boca Raton, chap 5


Reznik-Schuller HM (1983b) Carcinogens, the pulmonary endocrine cell, and lung cancer. In: Becker K, Gazdar A (eds) Proceedings, The endocrine lung in health and disease. Saunders, Philadelphia

Reznik-SchUller H, Mohr U (1974) Investigations on the carcinogenic burden by air pollution in man. X. Morphological changes of the tracheal epithelium in Syrian golden hamsters during the first 20 weeks of benzo( ajpyrene instillation: an ultrastructural study. Zentralbl Bakteriol (Orig B) 159: 503-525

Reznik-Schuller H, Mohr U (1975) Investigations on the carcinogenic burden by air pollution in man. XII. Early pathological alterations of the bronchial epithelium in Syrian golden hamsters after intratracheal instillation of benzo(a)pyrene. Zentralbl Bakteriol (Orig B) 160: 108-129

Reznik-Schuller H, Reznik G (1979) Experimental pulmonary carcinogenesis. Int Rev Exp Pathol 20: 211-281

Saffiotti U (1970) Experimental respiratory tract carcinogenesis and its relation to inhalation exposures. In: Hanna MG, Nettesheim P, Gilbert JR (eds) Inhalation carcinogenesis, AEC Symposium Series No 18 (CONF-691001). USAEC Div Tech Info Ext, Oak Ridge TN, pp 27-54

Saffiotti U, Cefis F, Kolb LH (1964) Bronchogenic carcinoma induction by particulate carcinogens. Proc Am Assoc Cancer Res 5: 55

Saffiotti U, Cefis F, Shubik P (1966) Histopathology and histogenesis of lung cancer induced in hamsters by carcinogens carried by dust particles. In: Severi L (ed) Lung tumours in animals. Div Cancer Research, University of Perugia, Perugia, pp 537-546

Saffiotti U, Montesano ~ Sellakumar AR, Kaufman DG (1972a) Respiratory tract carcinogenesis induced in hamsters by different dose levels ofbenzo(ajpyrene and ferric oxide. JNCI 49: 1199-1204

Saffiotti U, Montesano ~ Sellakumar A~ Cefis F, Kaufman DG (1972b) Respiratory tract carcinogenesis in hamsters induced by different numbers of administrations ofbenzo( ajpyrene and ferric oxide. Cancer Res 32: 1073-1081

Schreiber H, Saccomanno G, Martin DH, Brennan L (1974) Sequential cytological changes during development of respiratory tract tumors induced in hamsters by benzo(ajpyrene-ferric oxide. Cancer Res 34: 689-698

124 Gary A. Boorman

Squamous Cell Carcinoma, Lung, Rat

Gary A. Boorman


Gross Appearance

The gross appearance of spontaneous squamous cell carcinomas of the rat lung has apparently not been described in the literature. Squamous cell carcinomas induced in the rat lung by polycyclic hydrocarbons are seen grossly as solid masses containing central areas of friable material (keratin) (Shabad and Pylev 1970). Naturally occurring squamous cell carcinoma in man tends to arise in the hilar region and is thus more centrally located than other tumor types, which may arise in the periphery of the lung (WHO 1982). Induced squamous cell carcinomas of the rat lung often arise at the site of application of the carcinogen. A site predilection for the naturally occurring squamous cell carcinoma of the rat lung has not been reported.


The spontaneous squamous cell carcinomas found in the lung of the NCIINTP control F344 rats are composed almost entirely of squamous epithelial cells (Fig. 161). The tumor cells often show little atypia (Fig. 162), produce keratin, and show orderly differentiation (Fig. 163). Their malignant nature is evident by invasive growth into the surrounding lung parenchyma and one of the three naturally occurring tumors had metastasized to the mediastinum (Fig. 164). Induced squamous cell carcinomas of the rat lung appear more anaplastic. Microscopically the tumors contain both well-differentiated squamous epithelium and areas of atypical polymorphic cells with hyperchromatic nuclei and numerous mitotic figures (Shabad and Pylev 1970). Both naturally occurring and induced squamous cell carcinomas of the rat lung frequently have a marked scirrhous response (Kuschner and Laskin 1970; Shabad and Pylev 1970).

Ultrastructure

A description of the ultrastructural features of the spontaneous squamous cell carcinoma of the rat lung was not found in a review of the literature. In squamous cell carcinomas induced in the lung of the rat by N-nitrosoheptamethyleneimine, ultrastructural features include the formation of tonofilaments, keratohyalin, and keratin with invasive growth into the adjacent lung parenchyma (Reznik-Schuller and Gregg 1981).


Squamous cell carcinoma must be differentiated from squamous cell hyperplasia and also from bronchial alveolar carcinoma containing areas of squamous metaplasia. Rats with vitamin A-deficient diet and/or chronic murine mycoplasmosis may have extensive areas of squamous metaplasia and hyperplasia which could be confusing (Kuschner and Laskin 1970). Areas of squamous metaplasia have an orderly progression of differentiation from a layer of basal cells undergoing keratinization, have less atypia, and do not disrupt or invade normal pulmonary structures as will squamous cell carcinomas. The marked scirrhous response found in many squamous cell carcinomas is a useful feature in distinguishing the tumor from metaplasia. Distant metastases or invasion confirm the malignant nature of the lesion. Bronchiolar/alveolar carcinomas usually have a glandular pattern and are clearly different. In man, less well-differentiated adenocarcinomas

Fig.161 (Upper left). Squamous cell carcinoma, rat lung. ~ Note abundant keratin production. Hand E, x 80

Fig.162 (Upper right). Squamous cell carcinoma, rat lung. Note little cellular atypia and minimal keratin production. Hand E, x 160

Fig.163 (Lower left). Squamous cell carcinoma, rat lung. Orderly progression of differentiation is evident from basal layer to central area of keratin. Hand E, x 300

Fig. 164 (Lower right). Metastasis of pulmonary squamous cell carcinoma to mediastinum. Cells are less differentiated and mitotic figures are frequent. Hand E, x 350

Squamous Cell Carcinoma, Lung, Rat 125

126 Gary A. Boorman

can be distinguished from squamous cell carcinomas by different immunohistochemical patterns of keratin staining (Said et al. 1983). In the rat, well-differentiated bronchiolar/alveolar carcinomas may show areas of squamous cell differentiation. Since bronchiolar/alveolar carcinomas rise from different cell types in the periphery of the lung, it is crucial to separate them from the squamous cell carcinomas for proper interpretation of long-term toxicologic studies. Squamous cell carcinomas may be more central in location and should be composed entirely of squamous cells. Tumors showing a glandular or alveolar pattern mixed with squamous areas would be more properly diagnosed as bronchiolar/alveolar carcinoma with squamous differentiation.

Biologic Features

Induced squamous cell carcinomas of the rat appear to begin with basal cell hyperplasia in the airways. Bundles of tonofilaments are later found in the cells in the hyperplastic areas, suggesting squamous cell metaplasia. The squamous cell carcinomas later develop in the areas of metaplasia (Reznik-Schuller and Gregg 1981). Several studies on radiation-induced squamous cell carcinoma in the rat lung also showed progressive stages from squamous metaplasia to carcinoma (Kuschner and Laskin 1970). Squamous metaplasia of the respiratory epithelium precedes or may be a "precondition" for squamous cell carcinoma in man (Grundmann 1983; Saccomanno et al. 1970). Since this tumor type is one of the most frequent in man, several models have been developed to produce squamous cell carcinomas in the rat. Placement of a thread laden with methylcholanthrenes in the lung, intratracheal instillation of polycyclic hydrocarbons with absorbents such as India ink powder and carbon black, and implan-

tation of intrabronchial pellets containing polycyclic hydrocarbons all produce squamous cell carcinomas in the rat (Deutsch-Wenzel et al. 1983; Kuschner and Laskin 1970; Shabad and Pylev 1970). The induced tumors in the rat simulate bronchogenic carcinoma in man, arising from bronchial epithelium, being locally invasive, and having a tendency to metastasize to hilar lymph nodes and kidneys (Kuschner and Laskin 1970). In contrast to induced mouse pulmonary tumors, which are reported to be weakly immunogenic (Yuhas et al. 1975; Pasternak et al. 1966), squamous cell carcinomas induced in the rat respiratory tract are capable of eliciting both cellular and humoral response in immunized isogenic recipients (Jamasbi et al. 1978). In a review of the literature, most articles on naturally occurring tumors of various rat strains reported no squamous cell carcinomas of the lung. A few large series did contain one or more squamous cell carcinomas, as shown in Table 8. In 2-year studies squamous cell carcinomas of the lung were not found in female rats and occurred at a very low incidence in males (0.1 %-0.2%). In rats allowed to complete their life span a slightly higher incidence was found in males (0.6%) and one squamous cell carcinoma was found in a female rat (Table 8). There is no evidence in this series of any strain differences in incidence - apparently, they are very uncommon in most rat strains.


Squamous cell carcinoma is a very uncommon type of pulmonary neoplasm in the rat, as opposed to man, in whom this is the most frequent type of tumor (WHO 1982; Rothschild et al. 1982). In the dog and cat squamous cell carcinomas account for 6% and 12% of the lung tumors respectively (Moulton et al. 1981). The induced

Table S.N aturally occurring squamous cell carcinoma in the rat lung

Strain Age" Incidenceb

Males Females

Wi star 124 weeks 1/472 (0.2) 0/457 Osbome-Mendel 126 weeks 1/975 (0.1) 0/970 F344 116 weeks 312305 (0.1) 012356 F344 Life span 3/529 (0.6) 1/529 (0.2)

" All reports include animals dying spontaneously: age given is the age at time of death b Incidence equals no of animals with squamous cell carcinomal animals at risk (%)

Reference

Kroes et al. 1981 Goodman et al. 1980 Haseman et. al. 1984 Solleveld et al. 1984

Radiation-Induced Squamous Cell Carcinoma, Lung of Rodents 127

tumors in the rat are reported to have biologic characteristics analogous to those in man (Kuschner and Laskin 1970), but spontaneous tumors are rare and their biolgic characteristics have not been established.

References

Deutsch-Wenzel RP, Brune H, Grimmer G, Dettbarn G, Misfeld J (1983) Experimental studies in rat lungs on the carcinogenicity and dose-response relationship of eight frequently occurring environmental polycyclic aromatic hydrocarbons. JNCI 71: 539-544

Goodman DG, Ward JM, Squire RA, Paxton MB, Reichardt WD, Chu KC, Linhart MS (1980) Neoplastic and nonneoplastic lesions in aging Osborne-Mendel rats. Toxicol Appl Pharmacol 55: 433-447

Grundmann E (1983) Classification and clinical consequences of precancerous lesions in the digestive and respiratory tracts. Acta Pathol Jpn 33: 195-217

Haseman JK, Huff JE, Boorman GA (1984) Use of his torical control data in carcinogenicity studies in rodents. Toxicol Pathol in press

Jamasbi RJ, Nettesheim P, Kennel SJ (1978) Demonstration of cellular and humoral immunity to transplantable carcinomas derived from the respiratory tract of rats. Cancer Res 38: 261-267

Kroes R, Garbis-Berkvens JM, de Vries T, van Nesselrooy HJ (1981) Histopathological profile of a Wistar rat stock including a survey of the literature. J Gerontol 36: 259-279

Kuschner M, Laskin S (1970) Pulmonary epithelial tumors and tumor-like proliferations in the rat. In: Nettesheim P, Hanna MG, Deatherage JW (eds) Morphology of experimental respiratory carcinogenesis, AEC Symposium Series 21. USAEC, Oak Ridge, pp 203-226

Moulton JE, von Tscharner C, Schneider R (1981) Classification of lung carcinomas in the dog and cat. Vet Pathol 18:513-528

Pasternak G, Hoffmann F, Graffi A (1966) Growth of diethylnitrosoamine-induced lung tumours in syngeneic mice specifically pretreated with x-ray killed tumour tissue. Folia Bioi (Praha) 12: 299-304

Reznik-Schuller HM, Gregg M (1981) Pathogenesis of lung tumors induced by n-nitrosoheptamethyleneimine in F344 rats. Virchows Arch [Pathol Anat] 393: 333-343

Rothschild H, Buechner H, Welsh R, Vial LJ, Weinberg R (1982) Histologic typing of lung cancer in Louisiana. Cancer 49: 1874-1877

Saccomanno G, Saunders RP, Archer VE, Auerbach 0, Brennan L (1970) Metaplasia to neoplasia. In: Nettesheim P, Hanna MG, Deatherage JW (eds) Morphology of experimental respiratory carcinogenesis, AEC Symposium Series 21. USAEC, Oak Ridge, pp 63-82

Said JW, Nash G, Banks-Schlegel S, Sassoon AF, Murakami S, Shintaku IP (1983) Keratin in human lung tumors: Patterns of localization of different-molecular-weight keratin proteins. Am J Pathol 113: 27 -32

Shabad LM, Pylev LN (1970) Morphological lesions in rat lungs induced by polycyclic hydrocarbons. In: Nettesheim P, Hanna MG, Deatherage JW (eds) Morphology of experimental respiratory carcinogenesis, AEC Symposium Series 21. USAEC, Oak Ridge, pp 227 -242

Solleveld HA, Haseman JK, McConnell EE (1984) Natural history of body weight gain, survival and neoplasia in the F 344 rat. JNCI 72: 929-940

WHO (1982) Histological typing of lung tumors. Neoplasma 29: 111-123

Yuhas JM, Pazmiiio NH, Wagner E (1975) Development of concomitant immunity in mice bearing the weakly immunogenic line 1 lung carcinoma. Cancer Res 35: 237-241

Radiation-Induced Squamous Cell Carcinoma, Lung of Rodents

Fletcher F. Hahn


Gross Appearance

Squamous cell carcinomas usually appear as spherical or multilobulated nodules with circumscribed borders (Fig.165). They may be solitary or, on occasion, several may be found in one lung. Solitary nodules may achieve a relatively large size and occupy much of the thorax. There is no predilection for anyone lung lobe. They are

found in the parenchyma and are rarely oriented around major airways. The nodules are frequently seen as gray-white through the pleura. The pleura may be elevated by the nodule in the deflated lung, but invasion of the pleura is rare. On the cut surface, the nodules are dry, gray-white, and generally firm. In welldifferentiated tumors, the cut surface may be dry and caseous.

128 Fletcher F. Hahn

Fig. 165. Squamous cell carcinoma in the rat, note spherical nature and circumscribed borders typical of these tumors. F344 rat 588 days after inhalation exposure to an alpha-emitter, mixed transuranic oxides


The well-differentiated forms of squamous cell carcinoma are easily recognized by their mimicking of squamous epithelium and the keratinization of the more differentiated layers (Fig. 166). Frequently, the carcinomas are multilobulated, consisting of nests of readily identifiable squamous epithelium, keratohyalin, and epithelial pearls. The epithelium is dysplastic and contains foci of anaplastic cells. These tumors can grow to occupy nearly the entire lung. In some cases, much of the tumor mass comprises necrotic keratinized cells sloughed from a thin border of keratinized squamous epithelium at the periphery of the mass (Fig.168). These tumors are usually solitary with a circumscribed border. Classification of some of these well-differentiated tumors as malignant is equivocal, but the masses do grow slowly, compressing surrounding parenchyma and compromising pulmonary function. None of the well-differentiated tumors metastasizes or invades the pleura. The poorly differentiated forms of squamous cell carcinoma are still readily recognizable by their

individual cell keratinization (Fig. 167). Small nests of epithelial cells are formed, but there is no semblance of normal maturation of the epithelium. At the periphery of the nests, there is usually a layer of large anaplastic cells that may have a small amount of eosinophilic cytoplasm. Toward the center of the nest, the cells usually have more eosinophilic cytoplasm, indicative of individual cell keratinization (Fig. 169). Sometimes there is a fibrous stroma, but the tumors are usually not sclerosing. These tumors grow extensively within the lung and are locally invasive, but rarely metastasize or invade the pleura. Metastasis, when it occurs, is usually confined to the thorax and local lymph nodes. The invasiveness of these tumors is illustrated by their growth into pulmonary veins, which has been noted in both rats and mice. The invasive tissue is usually poorly differentiated (Fig. 170) but can be well differentiated.

Ultrastructure

The ultrastructure of these tumors has not been studied in rodents. However, ultrastructural characterization is not required for diagnosis because of the typical squamous characteristics of even the poorly differentiated tumors.


Grossly, squamous cell carcinomas must be differentiated from other lung tumors and from abscesses. The poorly differentiated tumors may appear grossly like adenocarcinomas of the lung. The squamous cell carcinomas are generally nodular, whereas the adenocarcinomas tend to fill the whole lobe of the lung. The well-differentiated squamous cell carcinomas may appear grossly like benign tumors or abscesses of the lung. Generally, the cut surface of the squamous cell carcinoma has a drier center with a lamellated gray appearance. Abscesses generally have a soft center with homogeneous appearance. Microscopically, squamous cell carcinomas must be differentiated from squamous metaplasia, squamous papillomas, and adenocarcinomas. Squamous metaplasia can be quite extensive in

Fig. 166 (Upper left). Well-differentiated squamous cell car- t> cinoma in the lung of an F 344 rat 588 days after inhalation exposure to a beta-emitter, 144Ce02. Two small foci of keratin formation are present. Hand E, x 100


Fig.167 (Lower left). Poorly differentiated squamous cell carcinoma in the lung of a Syrian hamster 398 days after inhalation of 144Ce02. Many individual cells have abundant cytoplasm with keratin. Hand E, x 100

Fig.168 (Upper right). Well-differentiated squamous cell carcinoma in the lung of an F 344 rat 453 days after inhalation exposure to mixed transuranic oxides. The periphery of the tumor is well differentiated and has produced

.. . .

~;' :. ,.: ... "',. ". t.,

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~ •. :.'" !' I ... : .. , ... • '. • ~ .' \ !.I~

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abundant keratin that is accumulated in the center. The tumor has compressed the surrounding normal lung parenchyma. Hand E, x 63

Fig. 169 (Lower right). Poorly differentiated squamous cell carcinoma in the lung of an F 344 rat 557 days after inhalation exposure to mixed transuranic oxides. Small nests are present, with a distinct peripheral layer of cells and a central accumulation of large cells with abundant eosinophilic cytoplasm. Hand E, x 100


Fig. 170. Growth of a poorly differentiated squamous cell carcinoma into a pulmonary vein of a C 57 BL/6J mouse 295 days after inhalation exposure to 144Ce02. Such invasion of vessels may be seen, but metastasis of radiation-induced squamous cell carcinomas outside the thoracic cavity in rodents is rare. Hand E, x 100

the lung, particularly in mice. Metaplasia is usually associated with bronchioles and alveolar ducts, but may extend to peripheral alveoli. Occasionally, metaplasia occurs solely in the alveoli, usually in association with focal scars or particulates in the alveoli. Metaplasia is well differentiated, especially in rats, and usually conforms to the normal alveolar architecture of the lung. Some well-differentiated squamous tumors in rats are large nodules with a well-differentiated wall of squamous epithelium and a center filled with keratin debris. These have been classified as endophytic papillomas. Histologically, they frequently resemble epidermal cysts with little papillary differentiation. They may occur as solitary nodules, but frequently malignant foci or masses are associated with the papillomas, indicating that malignant change occurs from the papillomas. Poorly differentiated squamous cell carcinomas may resemble adenocarcinomas, but individual squamous cells generally have a more abundant, pale eosinophilic cytoplasm with an angular outline.

Biologic Features

Rodents have been used extensively in studies of radiation carcinogenesis of the lung using internally deposited alpha- or beta-emitters or external irradiation. Modes of exposure have been inhalation, intratracheal instillation, or pellet implantation of radionuclides, or external thoracic irradiation. These different modes may affect the type of neoplasia or incidence of neoplasia. A good review of radiation carcinogenesis in the respiratory tract has been published by Kennedy and Little (1978). Table 9 gives results reported from studies with radioactive materials in rats. Squamous cell carcinomas are the most common radiation-induced tumors in rats exposed to beta or gamma radiation. Adenocarcinomas, however, occur more frequently in rats that inhale alpha-emitting radionuclides. This higher incidence may relate to differences in radiation dose distribution in the lung or to differences in the route of administration, i. e., inhalation vs implantation or instillation. Radiation quality factors may also playa role. Masse (1980) has commented on the histogenesis of lung tumors induced in rats by inhalation of alpha-emitters. He does not state specifically from which studies the examined rats came, but a previous report from the same laboratory indicates that the histologic type of lung tumor was independent of the radionuclide inhaled: 244Cm, 241Am, 235Pu, 238Pu, or 239Pu (LaFuma et al. 1974). Masse reviewed 500 epithelial lung tumors and classified them according to the World Health Organization classification of human lung tumors. The five most common types were: epidermoid carcinoma (40%), bronchogenic adenocarcinoma (13%), bronchoalveolar carcinoma (36%), combined epidermoid and bronchogenic adenocarcinoma (5%), and large cell carcinoma (1.6%). Transmission electron microscopy was used to examine 34 of the tumors, but improved the diagnosis only in the case of large cell carcinomas. Sanders and Dagle (1974) noted a direct correlation between the degree of inflammation and sclerosis around a radioactive particle and squamous metaplasia in surrounding tissue, ftequently in subpleural regions. They envisioned progression from squamous metaplasia to squamous cell carcinoma. Masse (1980), however, did not find scarring and metaplasia to be a prerequisite for the development of such carcinomas. Table 10 was prepared from studies with radioactive materials in mice. Squamous cell carcinomas in mice are induced infrequently by irradiation.


Table 9. Lung tumor types in rats exposed to radioactive materials'

Compound Exposure Percent prevalenceb Range of doses to lung (rad) Squamous Adeno- Adenoma Other

Other

Internal emitters Alpha-emitters

Particulates 238Pu 239Pu 244cd.. or 253Es

Gases RnandRn daughters

Beta-emitters Implants 32p 90Sr 106Ru ' Particulates 35S, 144Ce Particulates 144Ce

External irradiation

Inhalation

Inhalation

Implant

Intratracheal instillation Inhalation

7 - 10 x 1Q3

38 - 92 x 10Z WLMc

0.34- 16 x 1<r

24 -200 x 104

0.3 - 74 x 1<r

cell carcinoma carcinomas

<1-9 7-36

56 3

17-58

13-23

14-36 1-8

<1-3

2

Hemangiosarcoma Fibrosarcoma Mesothelioma

Lymphosarcoma

Reticulosarcoma

X-rays Thoracic 0.3 - 0.5 x 1<r 10 43 5 Fibrosarcoma

• Compiled from Annals of ICRP (1980) and Gracey et al. (1979); ranges are the range of averages for all the studies on any particular group of compounds

b Percent prevalence = percentage of animals at risk that develop tumor type noted C WLM, working level month; although estimates vary, 1 WLM a.; 0.5-1 rad

Table 10. Lung tumor types in mice exposed to radioactive materials'

Compound Exposure Percent prevalenceb Range of doses to lung (rad) Squamous Adeno- Adenoma Other

Internal emitters Alpha-emitters

239PU02

Beta-emitters Particulates 106Ru02 Particulates 9OY,144Ce

Gamma-emitters 6OCo wire

External irradiation X-rays

Intratracheal instillation

Intratracheal

Inhalation

Implant

Thoracic

120- 4000

300- 9000

990-32000

9- 46x1<r

750- 2750

cell carcinoma carcinomas

5 400 Fibrosarcoma

4C 75c Lymphosarcoma

2 <1-3c 9-13c Hemangio-sarcoma Fibrosarcoma

10

23c

• Compiled from Annals of ICRP (1980), Hahn et al. (1980) and Lundgren et al. (1981); ranges are the range of averages for all the studies on any given group of compounds

b Percent prevalence = percentage of animals at risk that develop tumor type noted c Increase over control


Table 11. Lung tumor types in Syrian hamsters exposed to radioactive materials"

Compound

Alpha-emitters Particulates 21OpO, Fe203 Particulates 238Pu, 239Pu Gases Rn and Rn daughters

Beta-emitters Particulates 144Ce02

Exposure mode

Intratracheal instillation Inhalation

Inhalation

Inhalation

Range of doses to lung (rad)

300-5000

66-9000

85- 120 x 102 WLMC

60- 500 x 102

Percent prevalenceb

Squamous Adeno- Adeno- Adenoma cell carcinoma squamous carcinomas carcinoma

3 97

2 <1-14 <1-21

2

3 2

a Compiled from Annals of ICRP (1980), Cross et al. (1981), Lundgren et al. (1982), Lundgren et al. (1983) and Thomas et al. (1981) - ranges are the range of averages for all the studies on any given group of compounds

h Percent prevalence = percentage of animals at risk that develop tumor type noted C WLM, working level month; although estimates vary, 1 WLM ~ 0.5-1 rad

Table 12. Comparison of squamous cell carcinoma characteristics in laboratory rodents and humans exposed to radiation

Characteristic Rats Mice Syrian Mana hamsters

Prevalence <1%-60% 0-10% 2%-3% 3% Differentiation Good Moderate Poor Poor Metastasis Rare Rare Rare Frequent Site of origin Small Small Small Large

airways, airways, airways, airways alveoli alveoli alveoli

a Based on data from uranium miners (Horacek et al. 1977 and Kunz et al. 1979)

The occurrence of these tumors in any study seems related to high radiation dose to the lung, for example with the 60Co wire implants. Table 11 contains results reported from studies with radioactive materials in Syrian hamsters. It is difficult to induce lung tumors in Syrian hamsters with radiation. The lone exception is the intratracheal instillation of 210pO and hematite. In this model, 15 weekly intratracheal instillations are given. Tumors can be induced in as little as 15 weeks, and by 1 year essentially all hamsters have tumors.


Squamous cell carcinomas of the lung of different species of laboratory rodents are histologically similar. There are differences, however, in several characteristics, as noted in Table 12. More differ-

ences are apparent between squamous cell carcinomas in laboratory rodents and those in man. Rarely are the tumors in laboratory rodents as poorly differentiated or aggressive as in man, where distant metastases are common. In man, squamous cell carcinomas most frequently originate in major airways; this is rare in rodents. This difference may be related to differences in the site at which the radiation dose is delivered.

Acknowledgements. Research was performed under U. S. Department of Energy Contract Number DE-AC04-76EV01013. Research was conducted using facilities fully accredited by the American Association for the Accreditation of Laboratory Animal Care.

References

Annals of the ICRP (1980) Biological effects of inhaled radionuclides. Pergamon, New York

Cross FT, Palmer RF, Busch RH, Filipy RE, Stuart BO (1981) Development of lesions in Syrian golden hamsters following exposure to radon daughters and uranium ore dust. Healthy Phys 41: 135-153

Gracey DR, Fish JE, Divertie MB (1979) Experimental squamous cell lung tumors in Sprague-Dawley and murine pneumonitis-free rats. Cancer 44: 558-603

Hahn FF, Lundgren DL, McClellan RO (1980) Repeated inhalation exposure of mice to 144Ce02. II. Biologic effects. Radiat Res 82: 123-137

HoracekJ, Placek V, SevcJ (1977) Histologic types of bronchogenic cancer in relation to different conditions of radiation exposure. Cancer 40: 832-835

Kennedy AR, Little JB (1978) Radiation carcinogenesis in the respiratory tract. In: Harris CC (ed) Pathogenesis and therapy of lung cancer. Dekker, New York

Kunz E, SevcJ, Placek V, Horacek J (1979) Lung cancerin men in relation to different time distribution of radiation exposure. Health Phys 36: 699-706

La Fuma J, Nenot JC, Morin M, Masse R, Metivier H, Nolibe D, Skupinski W (1974) Respiratory carcinogenesis in rats after inhalation of radioactive aerosols of actinides and lanthanides in various physiochemical forms.

Pleural Mesothelioma, Syrian Hamster 133

In: Karbe E, ParkJF (eds) Experimental lung cancer: carcinogenesis and bioassays. Springer, Berlin Heidelberg New York

Lundgren DL, Hahn FF, McClellan RO (1981) Toxicity of 90y in relatively insoluble fused aluminosilicate particles when inhaled by mice. Radiat Res 88: 510-523

Lundgren DL, Hahn FF, McClellan RO (1982) Effects of single and repeated inhalation exposure of Syrian hamster to aerosols of 144Ce02. Radiat Res 90: 374-394

Lundgren DL, Hahn FF, Rebar AH, McClellan RO (1983) Effects of the single or repeated inhalation exposure of Syrian hamsters to aerosols of 239PU02. Int J Radiat Bioi 43: 1-18

Masse R (1980) Histogenesis of lung tumors induced in rats by inhalation of alpha emitters: an overview. In: Sanders CL, Cross FT, Dagle GE, Mahaffey JA (eds) Pulmonary toxicology of respirable particles. DOE symposium series, 53 CONF-791 002. National Technical Information Service, Springfield

Sanders CL, Dagle GE (1974) Studies of pulmonary carcinogenesis in rodents following inhalation of transuranic compounds. In: Karbe E, ParkJF (eds) Experimental lung cancer: carcinogenesis and bioassays. Springer, Berlin Heidelberg New York

Thomas RG, Drake GA, London JE, Anderson EC, PrineJR, SmithDM (1981) Pulmonary tumours in Syrian hamsters following inhalation of 239Pu02. Int J Radiat Bioi 40: 605-611

Pleural Mesothelioma, Syrian Hamster

Antonio Cardesa and Josep A. Bombi

Synonyms: Malignant mesothelioma; mesothelial neoplasia.

Gross Appearance

Pleural mesotheliomas appear as whitish gray nodules, usually multiple and of firm consistency. When initially detected they form tiny masses of less than 1 mm in diameter, which progressively grow and fuse together to form larger nodules and plaques measuring up to 5 mm at the widest point. The development of these nodules takes place from the parietal as well as from the visceral pleura, extending along and covering the pleural surfaces. The pleural cavity may be filled by the growth of the mesothelioma; nevertheless, even in advanced lesions, gross invasion of the lung parenchyma is as a rule not evident. In some mesotheliomas the pleural thickening may be particularly striking at the interlobular fissures. These

lesions, when seen from the cut surface of the lung, may give a false impression of tumor invasion. Pleural effusions are occasionally present.


Three main histological types of mesotheliomas are recognized: epithelioid, fusocellular, and mixed. The epithelioid mesotheliomas are made up of large polyhedral cells, with abundant amphophilic cytoplasm and sharply defined cell contours. Their nuclei are vesiculated, rounded to oval in shape, with the chromatin mainly distributed adjacent to the nuclear membrane or attached to it. Frequently, epithelioid mesotheliomas give rise to the development of papillary formations (Figs. 171 and 172), which are supported by thin stalks of connective tissue. In other cases, epithelioid mesotheliomas are manifested initially in nodular forms (Fig. 173). Occasionally,

134 Antonio Cardesa and losep A. Bombi

<l Fig.171 (Upper left). Papillary mesothelioma, Syrian hamster, at early stage of development, growing at the pleural surface. Hand E, x 100

Fig. 172 (Upper right). Papillary mesothelioma, Syrian hamster. Epithelioid cells with mitotic figures. Hand E, x 400

Fig.173 (Lower left). Mesothelioma, hamster, attached to the pleural surface. Hand E, x 100

Fig.174 (Lower right). Epithelioid mesothelioma, Syrian hamster, with spreading pattern. Hand E, x 100

Fig.175 (Above). Epithelioid mesothelioma, Syrian ham- C>

ster. Cells with eccentric nuclei and a lymphoplasmacytoid appearance. Hand E, x 400

Fig.176 (Lower left). Fusocellular mesothelioma, Syrian hamster. Note cells with spindle-shaped cytoplasm imitating the pattern of fibrosarcoma. Hand E, x 200

Fig.177 (Lower right). Fusocellular mesothelioma, Syrian hamster. Note spindle-shaped cells with nuclei elongated in the direction of the cellular axis. Hand E, x 400


136 Antonio Cardesa and Josep A. Bombi

the epithelioid type of mesothelioma may have a tubular or gland-like arrangement of complex spaces, which mimic the features of adenocarcinomas. In most instances, it is possible to see how these tumor cells originate from the mesothelial covering (Fig. 174); in other cases they seem to develop from the submesothelial layer. Epithelioid mesotheliomas may grow in the form of wide sheets. In many areas these are composed of compactly arranged cells, in others the cells may be loosely attached to one another or even be devoid of intercellular cohesiveness. The nuclei of these cells may be eccentrically located, sometimes conferring upon them a plasmacytoid resemblance (Fig. 175). The fusocellular type of mesothelioma is composed of cells with spindle-shaped cytoplasm, poorly defined cell contours, and oval nuclei elongated in the direction of the main cellular axis (Figs. 176 and 177). Fusocellular mesotheliomas are able to form collagen, imitating the histological pattern of fibrosarcomas. This resemblance is occasionally striking, since they may even adopt a herringbone structural arrangement. The cells fre-

Fig. 178. Epithelioid mesothelioma, Syrian hamster. Tumor cells within lymphatic vessels. Hand E, x 200

quently have atypical mitoses. Fusocellular mesotheliomas may also be arranged in intertwining or tangled strands, thus mimicking malignant fibrous histiocytomas. Mesotheliomas of the mixed pattern have areas with characteristic epithelioid features alternating with other areas in which the fusocellular pattern is quite evident. In the hamster, the type of mesothelioma most commonly observed is epithelioid. These epithelioid cells are PAS positive, except after diastase digestion, and positive as well for alcian blue, but the color fades after treatment with hyaluronidase. The mesothelioma cells do not seem to contain mucin.

Ultrastructure

The ultrastructural features of mesotheliomas have been reported in other laboratory rodents (Davis 1979; Kawai 1979) but we are not aware of reports on the ultrastructural features of pleural mesotheliomas in the hamster.


The main differential diagnoses include neoplasias metastatic to the pleura, particularly carcinoma and sarcoma. In these instances, the correct diagnosis should be based on finding a primary neoplasm with the histologic features of the tumor found in the pleura. In distinguishing epithelioid mesotheliomas from metastatic carcinomas, the presence or absence of mucin in the pleural tumors is helpful because this substance is not produced by mesotheliomas. Another diagnosis to be considered is malignant lymphoma extending through the mediastinum, lung, and pleura. The epithelioid cells of mesothelioma, particularly when they lack cohesiveness, may adopt plasmacytoid features that may mimic a plasmacytoma or lymphoplasmacytic lymphoma. The absence of lymphoma in other organs should argue against that diagnosis. Finally, malignant mesotheliomas should not be confused w;ith reactive fibrous pleural plaques, which are fibroblastic proliferations producing abundant collagen, usually in the vicinity of asbestos fibers.

Biologic Features

Natural History. Most mesotheliomas manifest their malignant behavior by superficial growth

along the pleura. Invasion of the underlying chest wall or lung parenchyma is usually minimal or discrete in comparison to the tendency for intracavitary growth. A mesothelioma tends to fill the pleural cavities, resulting in considerable thickening and even fusion of both pleural surfaces. This may cause compression of lung parenchyma, which is accentuated by the development of pleural effusions. In advanced cases, progressive invasion of mediastinum, pericardium, and diaphragm may occur. Lymphatic vascular invasion (Fig. 178), metastases to regional lymph nodes, or distant metastases should be regarded as exceptional findings.

Pathogenesis. Mesotheliomas are a well-recognized tumor entity and epithelioid mesotheliomas are histologically quite similar to the cells lining the mesothelial surfaces. In spite of this, controversy exists with regard to their histogenesis. Some investigators maintain that they arise from cells of the mesothelial lining, others believe that they originate from the underlying submesothelial cells (Alvarez-Fernandez and Diez-Nau 1979; Cardesa et al. 1980). In tHe hamster, due to the paucity of studies, there is not yet enough information available to add to knowledge on this unsettled issue.

Etiology. Mesotheliomas are produced by exposure to different kinds of asbestos and glass fibers.

Frequency, Natural Occurrence, and Experimental Induction. Mesotheliomas in the hamster are induced tumors. In a study of spontaneous tumors in two hamster colonies (Pour et al. 1976) no mesotheliomas were observed. Spontaneous mesotheliomas were not recorded in a wide review of the literature (Mohr 1982). Rabson et al. (1960) were able to induce mesotheliomas by treatment of hamsters with virus and asbestos. Smith et al. (1964, 1965) induced mesotheliomas by intrapleural injections of amosite into hamsters. Although Smith et al. (1970) induced pulmonary neoplasias and pleural mesotheliomas by the combined instillation of chrysotile and benzopyrene into the lower respiratory tract of hamsters, no tumors were found when the instillation was of chrysotile alone. More recently, F. Pott (personal communication) has been able to produce pleural mesotheliomas in hamsters by the inhalation of crocidolite and various types of glass fibers.


Comparison with Similar Lesions in Human and Other Species

From the morphological standpoint, mesotheliomas induced in the hamster have striking gross as well as microscopic similarities with their counterparts in man (Brenner et al. 1982) and in other laboratory species (Pott et al. 1976).

References

Alvarez-Fernandez E, Diez-Nau MD (1979) Malignant fibrosarcomatous mesothelioma and benign pleural fibroma (localized fibrous mesothelioma) in tissue culture: a comparison of the in vitro pattern of growth in relation to the cell of origin. Cancer 43: 1658-1663

Brenner J, Sordillo PP, Magill GB, Golbey RB (1982) Malignant mesothelioma of the pleura: review of 123 patients. Cancer 49: 2431-2435

Cardesa A, Alvarez T, Pott F, Huth F, Mohr U Sept (1980) Morphological patterns of mesotheliomas produced by intraperitoneal exposure to various fibres. Abstracts XIllth international congress International Academy of Pathology, p 277

Davis JM (1979) The histopathology and ultrastructure of pleural mesotheliomas produced in the rat by injections of crocidolite asbestos. Br J Exp Pathol 60: 642-652

Kawai TO (1979) Histopathological studies on experimentally induced pulmonary, pleural and peritoneal neoplasms in mice by intraperitoneal injection of chrysotile asbestos and N-methyl- N-nitrosourethane. Acta Pathol Jpn 29: 421-433

Mohr U (1982) Tumours of the respiratory tract. In: Turusov VS (ed) Pathology of tumours in laboratory animals, Vol III, Tumours of the hamster. IARC Sci Publ No 34, Lyon, pp115-145

Pott F, Dolgner R, Friedrichs KH, Huth F (1976) L'effect oncongene des poussieres fibreuses. Ann Anat Pathol (Paris) 21: 237-246

Pour P, Mohr U, Cardesa A, Althoff J, Kmoch N (1976) Spontaneous tumors and common diseases in two colonies of Syrian hamsters. II. Respiratory tract and digestive system. JNCI 56: 937-948

Rabson AS, Branigan WJ, Legallais FY (1960) Lung tumors produced by intratracheal inoculation of polyoma virus in Syrian hamsters. JNCI 25: 937 -965

Smith WE, Miller L, ChurgJ, SelikoffU (1964) Pleural reaction and mesothelioma in hamsters injected with asbestos. Proc Am Assoc Cancer Res 5: 9 (abstract #234)

Smith WE, Miller L, Churg J et al. (1965) Mesotheliomas in hamsters following intrapleural injection of asb~stos. J Mt Sinai Hosp (NY) 32: 1-8

Smith WE, Miller L, Churg J (1970) An experimental model for study of carcinogenesis in the respiratory tract. In: NettesheimP, HannaMG, DeatherageJW (eds) Morphology of experimental respiratory carcinogensis. AEC symposium series no 21. USAEC, Division of Technical Information Extension, Oak Ridge

138 Bernard Sass and Annabel G. Liebelt

Metastatic Tumors, Lung, Mouse

Bernard Sass and Annabel G. Liebelt

Synonym. Secondary tumors of the lungs.

The spread of cancers to distant organs and the subsequent development of new foci present major challenges in research on tumor progression. Reports of spontaneously occurring mouse mammary tumors metastasizing to the lung first appeared in the early years of this century. Experiments with induced hepatic tumors and several types of transplanted tumors were reported but the pathogenesis of metastasis was still not understood. Metastasis involves several steps: detachment of cancer cells, either singly or in clumps, from the primary tumor; invasion of and subsequent passage within the vascular system to distant sites in which adhesion to and invasion and penetration of the vessel wall occur; and infil-

Fig. 179. Large, multinodular metastasis in lung from spontaneous mammary tumor in strain RIll female mouse. x 15

tration and growth in the pulmonary tissues. These steps are at present being investigated and will result in a better understanding of the pathogenesis of metastasis.

Gross Appearance

There are few authoritative morphological descriptions of metastatic tumors of the lungs of mice (BorreI1903; Murray 1908; Ashburn 1937; Dunn 1945; Brooks 1970). Mouse lungs bearing metastases may have single or multiple nodular growths distributed in one or more lobes, and there is often bilateral involvement. Brooks (1970) described mammary tumor metastases as circumscribed groups of nodules, surrounded by narrow zones of compressed lung tissue. Tumor nodules may be found deep within the parenchyma of the lung as well as on the pleural surface. Stewart and associates (1970) described the gross appearance of alveologenic carcinomas as sharply circumscribed pearly white nodules which may project above the pleural surface of the lung. In the experience of the authors, metastatic mammary tumors are usually soft and friable, grayish white, and highly vascular, and have a raised nodular surface (Fig.179); metastases of mammary tumors with extensive squamous metaplasia or keratinization may appear pearly white. Hepatic tumor metastases are firm, round, pale browngray, and well circumscribed (Vesselinovitch et al. 1978). Metastases of primary tumors from connective tissues, cartilage, or bone, on the other hand, are usually dense, firm to very hard, relatively avascular, white, and shiny. Transplants of malignant melanoma may form metastases in the lungs as either pigmented (black, shiny, and rubbery) or nonpigmented (white, firm) tumors, depending on whether the transplant was pigmented or not (Figs.180 and 181). Although with most lymphoreticular neoplasms the lungs / are involved, the lungs often do not contain grossly visible discrete nodules but instead contain a diffuse infiltrate of tumor cells in the entire lung, causing a loss of normal pink color. Heston (1940), using a dissecting microscope, enumerated tumors in the lungs of mice. Several other methods for the detection and enumeration of lung tumors have been described. Wexler

(1966) devised a method for the identification of tumors utilizing the injection of India ink into the trachea of an anesthetized mouse; the ink is allowed to flow into the alveolar spaces by gravity. The dissected lungs are washed in tap water and then immersed in Fekete's solution (Fekete 1938). This solution bleaches the neoplastic tissue (Fig. 182), sharply outlining the gross tumors. Subsequently, the lobes of the lung are separated and sliced and the metastatic tumors are identified and counted. Histological identification and enumeration of metastatic tumors are improved by employing serial sectioning of the lung. Ashburn (1937) found 178 of 480 (37%) mammary-tumor-bearing mice to have gross metastases. He cited Murray (1908), who found 39.6% gross metastases in 68 tumor-bearing mice, Haaland (1911), who found 38% in 237, and Marsh (1929), who found 39.1% in 314. However, when Ashburn examined the 178 gross lesions, 31 of these (17.4%) were spurious metastases, that is, lung adenomas, lymphoid deposits, abscesses, foci of bronchopneumonia, or other circumscribed inflammatory lesions. Furthermore, when examining grossly negative lungs, Ashburn found 70 (14.6%) microscopic metastases. Murray (1908) and Marsh (1927) identified, by serial section techniques, metastases in eight of 16 and nine of 13 grossly normal lungs respectively. A study by Consolandi and associates (1958) revealed macroscopic pulmonary metastases microscopically confirmed in 34% of the cases and, in addition, 46% microscopic metastases in apparently normal lungs. The authors did not state which sectioning techniques were used. All of these studies point out the difficulty of relying onlyon data based on gross observations. Kyriazis et al. (1974) and Vesselinovitch et al. (1978) studied the percentage of pulmonary metastases from hepatic tumors induced by diethylnitrosamine (DEN). Their technique was to remove the lungs en bloc with the trachea, thymus, paratracheal lymph nodes, and thyroid glands, and fix these tissues in 10% neutral buffered formalin. The specimens, including the structures mentioned above, were processed whole, and 5-llm sections were cut at the long axis at two different levels, one of which passed through the hilar region. Kyriazis et al. (1974) found a metastatic percentage of 21.6% for animals receiving DEN at 1 day of age and 22.9% in animals receiving DEN at 15 days of age. Histologic examination of single, randomly selected lung sections yielded a

Metastatic Tumors, Lung, Mouse 139

metastatic percentage of only 4.3% for males and 0.0% for females. These studies demonstrate the importance of examining adequate sections of all lobes of the lung to obtain the exact number of tumor metastases.

Fig. 180 (Above). Pulmonary metastases (arrows) from subcutaneous transplant of melanotic melanoma in (BALBI c x DBAf)Fl hybrid mouse. Note a small amelanotic portion of the transplant (arrow). x 2

, Fig.181 (Below). Pulmonary metastases from subcutaneous transplant of amelanotic melanoma in (BALBI c x DBAf)Fl hybrid mouse. This transplant line was derived by selection of an amelanotic portion from the melanotic transplant line. x 2

140 Bernard Sass and Annabel G.Liebelt

1111111111111111111111111111111 Fig. 182. Lungs of strain RIll mouse with metastatic mammary tumor from a single mammary tumor. The lungs were distended with India ink; numerous pulmonary nodules are bleached white following exposure to Fekete's solution. x 2.5

Microscopic Features and Differential Diagnosis

One of the most important biologic criteria in the expression of primary malignant neoplasms is the ability of tumor cells to invade surrounding normal tissues, including the blood and lymph vessels. It is this vascular invasion that makes the first stage of metastasis, namely the formation of tumor cell emboli, a unique and essential prerequisite for the spread of malignant tumors (Willis 1973). Mouse tumors metastasizing to the lungs do so mainly by the hematogenous route (Ashburn 1937; Dunham and Stewart 1953; Consolandi et al. 1958; Kyriazis et al. 1974; Vesselinovich et al. 1978). There are three main anatomic sites in which tumor cells are found in the lungs. The first is within larger blood vessels in the form of tumor cell emboli or thrombi (Figs.183-185). Although tumor cell thrombi or emboli provide evidence of malignancy, they are not considered to be metastases (Willis 1973) until the constituent tumor cells penetrate vessel walls. The second site in the lungs is outside larger vessels but surrounding bronchi (Fig. 183) and within alveoli and alveolar capillaries. The third anatomic pattern which is common in metastatic tumors of the lungs of mice is that of disseminated small or large multiple foci in which the relationship to vessels is lost (Fig. 186).

The tumors invade veins rather than arteries, presumably because veins have thinner walls. Most blood-borne metastases are carried by the vena cava to the right heart and thence to the pulmonary artery. Tumor cells enclosed by vessel walls are considered to be emboli when they are nonadherent to vessel walls and thrombi when they are adherent. Tumor cell thrombi may contain other blood elements, such as leukocytes and thrombocytes. Not all tumor emboli or thrombi produce viable metastases (Fidler 1980). Those that do so may lodge in capillaries or larger vessels, the walls of which are penetrated by tumor cells. Having gained entry to pulmonary vessels, there are two main sites at which secondary tumors become established within the lung. The first, intraalveolar, is a frequent site of extension. In mice, alveolar architecture is lost early. Tumor cells invade alveolar spaces and fill and distend alveoli, the walls of which are destroyed by compression. The second site is interstitial extension, and includes invasion of lymphatics, veins, arteries, and bronchi; this is not often evident in mice, except those with primary lung tumors that spread within the lung. Tumors which establish in the lung by other than the hematogenous route have been reported by several authors (Furth 1946; Epstein 1966). These authors injected leukemia or carcinoma cells intratracheally, bringing about leukemia or carcinoma in the lung. There is a histological similarity between primary alveologenic (alveolar/bronchiolar) tumors and pulmonary metastases from adenocarcinomas of the mammary gland and from tumors of other or-

Fig.183 (Upper left). Lung, mouse, metastases from a sub- t> cutaneous sarcoma, not otherwise specified. Note peribronchial distribution of growths, upper left and center. A branch of the pulmonary artery (upper center) contains tumor cells. Hand E, x 54

Fig. 184 (Upper right). Lung, mouse, mammary tumor cell emboli in longitudinally sectioned pulmonary artery. The surrounding parenchyma is normal and the integrity of the vessel wall is intact. Hand E, x 80

Fig.185 (Lower left). Orbit, mouse, contents of the orbit with retina below, eyelid above and to left. Harderian gland to right and soft tissues between. The harderian gland is largely replaced by a carcinoma that has invaded a nearby vein (arrow). Hand E, x 54

Fig. 186 (Lower right). Lung, two large metastases of hepatocellular carcinoma at either end of the photomicrograph and a small one in the midsection. Note compression of alveoli and invasion of alveolar walls. Hand E, x 38



gan sites (kidneys, pancreas, uterus) which makes it difficult to identify the primary site (Reznik 1983). Other primary tumors of the mouse lung, such as squamous cell carcinoma, rarely occur spontaneously but have been induced by intratracheal instillation of methylcholanthrene. Alveologenic carcinoma develops in the alveolar walls from type II alveolar epithelial cells, which are cuboidal to columnar and are arranged in a glandular pattern often exhibiting papillary formations (Stewart et al. 1970, 1979; Stewart 1975). Adenomatosis (see chapter by Boorman, Bronchiolar/ Alveolar Hyperplasia) is a proliferative and inflammatory lesion which needs to be distinguished from alveologenic or metastatic tumors. Adenomatosis may be differentiated from alveologenic tumors by the following criteria: (a) the pleura is depressed, (b) the alveolar architecture is preserved, and ( c) mucus-containing ciliated or non ciliated columnar and cuboidal cells line the walls of the alveoli (Stewart et al. 1970). Adenomatosis may follow infection and recovery from Sendai virus. When extrapulmonary primary neoplasms occur singly, their metastases in the lung are readily identified provided that they reproduce the same cellular and architectural patterns as in the primary tumor. When, however, primary tumors of two or more different sites yield single or multiple pulmonary metastases, or if the pulmonary metastases differ in morphology from the primary tumor, it may be difficult to ascribe the origins of the metastases. In reticulum cell neoplasm type B and lymphocytic leukemia, nodular deposits may be found in the lungs of mice surrounding both vessels and bronchi. Reticulum cell neoplasm, type B, described by Dunn (1954) and Dunn and Deringer (1968), forms cuffs of tumor cells which surround bronchi and nearby vessels. Lungs of mice with lymphocytic leukemia, in addition, often contain intravascular plugs and deposits of neoplastic lymphocytes in alveolar capillaries. A differential diagnosis must be made distinguishing these patterns of hematopoietic tumors from peribronchial and perivascular cuffing due to proliferating macrophages and infiltration by lymphocytes following lesions of chronic pneumonia. Hepatocellular carcinoma and adenocarcinoma of the mammary gland are two tumors that frequently occur in inbred mice and commonly metastasize to the lung. The morphology of mammary gland tumors varies widely within and between strains; indeed, it varies between tumors

within one mouse and within individual tumors. Mammary tumors which metastasize to the lung usually have the same predominant histologic pattern as the parent tumor, and multiple tumors of different histologic types may produce metastases that correspond to the primary tumors (Fig. 187). Brooks (1970) reported that a group of metastatic nodules from mammary tumors may become surrounded by a zone of compact tissue containing smaller metastatic tumor nodules mixed with cells of lung origin. Pitelka et al. (1980a, b) examined 160 primary mammary tumors by both light and electron microscopy; 69 of these metastasized to the lung and the metastases were confirmed histologically (see also Ultrastructure). In describing the pulmonary metastases, the authors stated that "sections of large metastatic tumors often suggest a close resemblance to a primary tumor in the same mouse but almost as frequently do not." Otherwise, the morphology of the mammary epithelial cells in the primary and secondary sites was similar. Examination of serial paraffin and epon thick sections of grossly visible pulmonary metastases revealed tumor deposits within the arterioles accompanying bronchioles (Pitelka et al. 1980b). The tumor invaded the pulmonary connective tissue by penetrating the muscular and elastic layers of affected arterioles. Hepatocellular carcinomas induced by carcinogens are more likely to metastasize than spontaneous tumors (Figs.188 and 189) (Vesselinovitch et al. 1978). The increased frequency of metastasis of hepatocellular carcinoma is related to trabecular and undifferentiated patterns (see Biology section). Pulmonary metastases from liver tumors induced by DEN (Kyriazis et al. 1974; Vesselinovitch et al. 1978) were usually multiple and often of microscopic size, and closely resembled those of the primary tumor. There was the suggestion that isolated foci may have coalesced to form larger deposits. The alveolar capillaries contained tumor cell masses and the resultant growth occasionally distorted bronchiolar lumina (Fig. 188) and compressed the surrounding lung tissue (Fig. 189). The pulmonary metastases from hepatocellular carcinomas reduplicate the histologic pattern of the primary tumor. The rare hepatoblastoma can be differentiated from hepatocellular carcinoma by several chracteristics, including: (a) deep staining in hematoxylin and eosin preparations, (b) presence of spindle cells and rosettes, ( c) presence of peculiar "organoids," and (d) presence of hepatocellular carcinoma in the same section (Turusov and Takayama 1979).

Fig.187 (Upper left). Lung, mouse, metastatic mammary adenocarcinoma, type B compresses surrounding alveoli. Hand E, x 54

Fig. 188 (Upper right). Lung, mouse, large metastatic hepatocellular carcinoma that has elevated the pleura and compressed the lung parenchyma and wall of the alveolar duct. Hand E, x 54

Fig. 189 (Below). Higher magnification of section shown in Fig. 188. Hand E, x 220



<l Fig.190 (Above). Lung, mouse, metastasis of ovarian granulosa cell tumor that has replaced most of the pulmonary tissue of the apex of the lobe. The pleura is mineralized. H and E, x 54

Fig. 191 (Below). Higher magnification of section shown in Fig. 190. The neoplastic cells are arranged as small nests. H and E, x 130

Fig.192 (Upper left). Lung, mouse, metastasis of renal pel- ~ vic transitional cell carcinoma. The tumor cells are arranged as nests. Hand E, x 220

Fig.193 (Upper right). Lung, mouse, metastasis of renal adenocarcinoma composed of small, closely packed cells with indistinct borders arranged as sheets and nests. Hand E, x 130

Fig.194 (Lower left). Lung, mouse, metastasis of renal adenocarcinoma that has compressed and invaded alveoli. Glands are absent. The tumor cells have sharply defined borders, pale-staining cytoplasm, and eccentrically placed, often multiple, nuclei. Hand E, x 220

Fig.195 (Lower right). Lung, mouse, metastasis of harderian gland carcinoma. The tumor cells have pale-staining, often vacuolated cytoplasm. Hand E, x 220

Metastases of hepatocellular carcinomas must be differentiated from metastases of adrenal cortical carcinomas and from metastases of granulosa cell tumors. In metastases of adrenal cortical carcinomas and of granulosa cell tumors (Figs. 190 and 191) the cells tend to be arranged in groups invested by a fine fibrovascular stroma. Granulosa cell tumors with glomerate or tubular pattern may be differentiated from the metastases of adrenal cortical carcinoma. Two mice treated with 2-fluorenylacetamide had transitional cell carcinomas of the urinary bladder which metastasized. Both primary tumors had associated squamous cell metaplasia (Frith et al. 1981), but it was not clear whether the metastases had a squamous component. A renal pelvic transitional carcinoma produced metastases composed of nests (Fig. 192). Histologically, it may be difficult to dIstinguish metastases of renal adenocarcinomas (Figs.193 and 194) and ofharderian gland tumors (Fig. 195) from primary alveologenic tumors. Pulmonary metastases of renal adenocarcinomas may produce different patterns depending on the patterns of the primary tumor. In the BALB/c/Cf/Cd mouse strain, renal adenocarcinoma cells are small, have scant cytoplasm, and have poorly de-



Fig. 196 (Upper left). Lung, mouse, metastasis of malignant schwannoma in what remains of vessel wall. Tumor cells have poorly defined borders. Hand E, x 330

Fig.197 (Upper right). Lung, mouse, multiple t~rombi of malignant schwan noma adherent to wall of pulmonary vessel. Hand E, x 330

Fig. 198 (Below). Lung, mouse, pleural metastasis of osteosarcoma (A) and adjacent alveologenic carcinoma (B). H and E, x 130

fined borders (Fig. 193). In BALB/c mice the other type of renal adenocarcinoma induced by chemicals (Fig.194) is composed of tumor cells that are larger and have eccentric nuclei, abundant, almost clear cytoplasm, and clearly defined cell borders. Spontaneous harderian gland neoplasms in untreated mice, reported by Sheldon et al. (1983), were classified as adenocarcinoma or one of four different types of adenoma. Of the 30 mice with adenocarcinomas, the tumors of three metastasized to the lung and these had a histologic pattern similar to that of the primary tumor. The primary tumors of two of these three mice and of two other mice had spread to the periorbital tissues. The authors suggested that periorbital invasion precedes occurrence of distant metastases (Fig. 185). Primary tumors of harderian glands and their metastases (Fig.195) have the distinguishing feature that the tumor cells may contain intracytoplasmic vacuoles (Sheldon et al. 1983). Pulmonary metastases from sarcomas, especially those that are undifferentiated, form prominent cuffs around blood vessels and bronchi (Fig. 183). With hemangiosarcomas (hemangioendotheliomas) in the lung it may be difficult to determine whether they are primary or metastatic or represent one site of involement by a tumor of multicentric origin. Frith et al. (1981) assigned a metastatic origin to hemangiosarcomas in the lungs of seven mice treated with 2-fluorenylacetamide, on the basis of finding pulmonary emboli and miliary distribution of tumor. Malignant schwannomas, metastatic to the lung, consist almost wholly of Antoni type A tissue, namely interlacing parallel bundles and fibrils (Fig. 196). Lacking were other features of primary schwannoma, including palisading of the nuclei, Verocay bodies, cysts, and lipoid-containing pseudoxanthomatous cells. The pulmonary vessels contained mUltiple adherent thrombi (Fig. 197). The pulmonary metastases of osteosarcomas exhibit all characteristics of the parent tumor, including bone, osteoid, and spindle cells or undifferentiated cells. Among the sections filed in the Registry of Experimental Cancers are those of a mouse with a primary osteosarcoma of bone that had metastasized to the lung, in which there was also a primary alveologenic tumor. The two types of tumor were intermingled (Fig. 198).


Ultrastructure

Examination by electron microscopy of type B mammary tumor metastases in the lung by Brooks (1970) confirmed the acinar pattern of the tumor cells and revealed virus particles budding from cell surfaces, within the gland lumens, and also in some type B (II) cells of the pulmonary alveoli. Wide spaces containing fibrillar material were present at intervals between cells. The mammary tumor cells in the lung were separated from the alveolar airspace by the following: (a) a thick basallamina of the mammary tumor cells, (b) a loose connective tissue space containing fibroblasts and collagen fibrils, (c) the basal lamina of the alveolar epithelial cells, and (d) the alveolar cells themselves. Hyperplastic type B (II) pulmonary alveolar cells arranged in the form of acini were present in proximity to the metastatic nodules. Cystic spaces lined by mammary tumor cells in some metastatic nodules contained both membranous and fibrillar material. This material was believed by Brooks (1970) to have been synthesized by the tumor cells. Pitelka et al. (1980a, b) compared the ultrastructure of the junctions of the epithelial cells and basal lamina of normal mammary gland tissues, hyperplastic alveolar nodules (HANs), primary mammary carcinomas, and their pulmonary metastases. Tight junctions, characteristic of epithelial tissues and of normal nonlactating glands, were demonstrated between cells of the neoplastic glands. A feature of the tumors, not present in normal mammary tissue, was the presence of microlumina in otherwise solid deposits. These microlumina were characterized by tight junctions and apical microvilli. Also present in the tumor tissue at varying distances from tight junction belts but not in normal tissue were common macular tight junctions. The authors concluded that since the neoplastic cells have tight junctions, it cannot be proposed that a generalized reduction of adhesive incapability attributable to faculty junctions is a necessary characteristic of malignant epithelial cells. Normal basal lamina functions as a barrier between the normal adult epithelium which secretes it and the connective tissue; it also serves as a barrier limiting access of macromolecules (Fawcett 1981). Thus it is normally not penetrated except by migrating leukocytes and macrophages. Pitelka et al. (1980b) examined the basal lamina ofprimary and metastatic mammary tumors, and observed that hypertrophy of the basal lamina was the most common structural abnormality of the


Table 13. Pulmonary metastases from tumors in untreated mice

Organ of origin Diagnosis No. of mice Per- Sex Strain Reference with cent metastasis No. of mice with tumors

Liver Hepatoma 1-2 Slye et al. (1915) Hepatoma 11 17 6 M,F CBA Gorer (1940) Hepatoma 11 97 1 M CF-l Turusov et al.

(1973 b) Hepatoblastoma 01 3 0 M CF-l Turusov et al.

(1973 b) Hepatocellular 43/349 12 M B6C3HF1 Ward et al. (1979) carcinoma 71 58 12 F B6C3HF1 Ward et al. (1979)

51 50 10 M B6C3HF1 National Toxicology Program (1982 a)

01 50 0 F B6C3HF1 National Toxicology Program (1982a)

Mammary Carcinoma nos. 1041273 38 Haaland (1911) gland 261 68 40 Murray (1908)

40 Marsh (1927) 62 (following massage) Marsh (1929)

123/314 39 217/480 45 Infrequent

16 69/160 43

78/464 17 391 80 49 65/169 39 161 78 20 231 58 40 181 61 30

A, B, mixed AB 401 51 78 161 28 57 121 26 46

Carcinoma, nos. 121 19 63

11 6 17 Harderian Adenocarcinoma 31 30 10 gland

nos, not otherwise specified

tumor cells; interruptions were rare, except where necrosis occurred. The hypertrophy was evident as extensive folding, mUltiple layering or irregularly increased thickness and could be found: (a) in primary tumors, between stroma and mammary tumor epithelial cells, (b) between invasive primary tumors and surrounding connective tissue, and (c) in metastases between neoplastic mammary epithelium and tissues of the lung. The authors (Pitelka et al. 1980a) concluded that

Marsh (1929) NIH white Ashburn (1937) AIBrA van der Valk (1981) BALB/cfC3H van der Valk (1981) C3H,C3Hf Pitelka et al. (1980a) BALB/cfC3H BALB/cNIV A., GR, and RIll RIll Liebelt et al. (1968) C3H Liebelt et al. (1968) A Liebelt et al. (1968) DBAl2 Liebelt et al. (1968) RIIIllmr (breeder) Liebelt et al. (1981) RIIIllmr (virgin) Liebelt et al. (1981) BALB/cflC3H/Cb/Se Consolandi et al. RIIIIDmlSe (1958) C3H/Cb/Se Consolandi et al.

(1958) BALB/cfC3H Squartini and

Bistocchi (1977) BALBI cfRIII BALB/C, C3H/He Sheldon et al. (1983) and C57BLl6J

neoplastic mammary epithelium maintains an effective basal lamina barrier while invading nonepithelial tissues. Intravascular metastases contained basal lamina interposed between tumor cells and pulmonary vascular endothelium, or if such epithelium ruptured, the basal lamina was found between the tumor cells and the elastica. Following rupture of the arteriolar wall, basal lamina was found between tumor cells and pulmonary connective tissue.


Table 14. Pulmonary metastases from induced tumors in mice

Tissue of Diagnosis No. of mice Per- Treatment ~ex Strain Reference origin with cent

metastasis No. of mice with tumors

Liver Trabecular 221 102 22 DEN at 1 day of age M,F C57BLI Kyriazis et al. (1974) carcinoma 6Jx Trabecular 271 118 23 DEN at 15 days of age M,F C3HeBI Kyriazis et al. (1974) carcinoma FEJ hybrid Trabecular 2661 733 36 One of: BaP, ENU, B6C3F1 Vesselinovitch et al. carcinoma benzidine 2-HCl (1978) Trabecular 52/1076 5 2-FAA in Purina meal F BALBI Frith et al. (1981) carcinoma cStCr!

fC3H/Nctr Hepatoblastoma 0 Controls M,F CF-1 Turusov et al. (1973 a) Hepatoblastoma 4 DDT 2 ppm M,F CF-1 Turusov et al. (1973 a) Hepatoblastoma 0 DDT10ppm M,F CF-1 Turusov et al. (1973 a) Hepatoblastoma 4 DDT 50 ppm M,F CF-1 Turusov et al. (1973 a) Hepatoblastoma 11 DDT 250 ppm M,F CF-1 Turusov et al. (1973 a) Hepatoma 1 Controls M,F CF-1 Turusov et al. (1973 a) Hepatoma 2 DDT 2 ppm M,F CF-l Turusov et al. (1973 a) Hepatoma 2 DDT 10 ppm M,F CF-l Turusov et al. (1973 a) Hepatoma 1 DDT 50 ppm M,F CF-l Turusov et al. (1973 a) Hepatoma 1 DDT 250 ppm M,F CF-1 Turusov et al. (1973 a) Trabecular M,F B6C3F1 National Toxicology carcinoma Program (1982 a) Trabecular 41 49 8 DEHA 12000ppm M B6C3F1 National Toxicology carconoma Program (1982a) Trabecular 51 49 10 DEHA 25000 ppm M B6C3F1 National Toxicology carcinoma Program (1982a) Trabecular 61 49 12 DEHA 12000 ppm F B6C3F1 National Toxicology carcinoma Program (1982a) Trabecular 51 48 10 DEHA 25000 ppm F B6C3F1 National Toxicology carcinoma Program (1982a) Hepatocellular B6C3F1 National Toxicology carcinoma Program (1982b) Hepatocellular 71 49 14 DEHP 12000 ppm M B6C3F1 National Toxicology carcinoma Program (1982b) Hepatocellular 51 50 10 DEHP 25000 ppm M B6C3F1 National Toxicology carcinoma Program (1982b) Hepatocellular 11 50 2 DEHP 12000 ppm F B6C3F1 National Toxicology carcinoma Program (1982b) Hepatocellular 71 50 14 D EHP 25000 ppm F B6C3F1 National Toxicology carcinoma Program (1982b) Trabecular 21 8 25 2,7-FAA 250 ppm for M (DBAl2 Takayama (1968) carcinoma 3-5mo xC57BL)Flo

(DBF1)

Trabecular 31 10 30 F (DBAl2 Takayama (1968) carcinoma xC57BL)Flo

(DBF1)

Trabecular 21 15 14 M (DBAl2 Takayama (1968) carcinoma xC57BL)Flo

(DBF1)

Trabecular 51 16 31 F (DBA12 Takayama (1968) carcinoma xC57BL)Flo

(DBF1)

Trabecular 13 Dieldrin M,F CF-1 Thorpe and Walker carcinoma (1973)


Table 14 (continued)

Tissue of Diagnosis No. of mice Per- Treatment Sex Strain Reference origin with cent

metastasis No. of mice with tumors

Trabecular 61 Dieldrin M,F CF-1 cited by Vesselinovitch et carcinoma al. (1978) Trabecular 21 9 22 Nafenopen M Acatal- Reddy et al. (1976) carcinoma asemic Trabecular 31 12 25 Nafenopen F CSb Reddy et al. (1976) carcinoma

Mammary Tumors' 491 517 10 2-FAA F BALBI Frith et al. (1981) gland cStCrl

Other tumors fC3H/Nctr Osteogenic 61 13 46 2-FAA F BALBI Frith et al. (1981) sarcomab cStCrl

fC3H/Nctr Renal 41 20 20 2-FAA F BALBI Frith et al. (1981) carcinoma cStCrl

fC3H/Nctr Fibrosar- 31 18 17 2-FAA F BALBI Frith et al. (1981) comab cStCrl

fC3H/Nctr Myoepithe1io- 161 153 11 2-FAA F BALBI Frith et al. (1981) rna cStCrl

fC3H/Nctr Undifferen- 41 72 6 2-FAA F BALBI Frith et al. (1981) tiated cStCrl sarcomab fC3H/Nctr Leiomyosar- 11 36 3 2-FAA F BALBI Frith et al. (1981) comab cStCrl

fC3H/Nctr Granulosa cell 41 197 2 2-FAA F BALBI Frith et al. (1981) tumor cStCrl

fC3H/Nctr Adrenocorti- 31 181 2 2-FAA F BALBI Frith et al. (1981) calcarcinoma cStCrl

fC3H/Nctr Ovary Spleen Angiosarcoma 71 452 2 2-FAA F BALBI Frith et al. (1981) Skin cStCrl

fC3H/Nctr Squamous cell 11 76 1 2-FAA F BALBI Frith et al. (1981) carcinomab cStCrl

fC3H/Nctr Harderian Tumor 2312400 1 2-FAA F BALBI Frith et al. (1981) gland cStCrl

fC3H/Nctr Urinary Transitional cell 21 722 <1 2-FAA F BALBI Frith et al. (1981) bladder carcinoma cStCrl

fC3H/Nctr

DEN, diethylnitrosamine; BaP, benzo (a) pyrene; ENU, ethylnitrosourea; benzidine, p-diaminodiphenyl; '2-FAA, N-2-fluorenylacetamide; 2,7-FAA, N, N' -2, 7 -fluorenylenebisacetamide; DDT, 1,1, 1-trichloro-2,2-bis (p-chlorophenyl)ethane; DEHA, di (2-ethylhexyl)-adipate; DEHP, di (2-ethylhexyl)-phthalate; Dieldrin, 1,2,3,4,10,10-hexachloro-6,7-epoxy-1,4,4 a,5,6,7 ,8,8 a-octohydroendo-exo-1,4 :5,8-dimethanonaphthalene; Najenopen, 2-methyl-2- [P-(1,2,3,4-tetrahydro-1-naphthyl) phenoxy )proprionic acid • 4% adenocarcinoma type A, 73% adenocarcinoma type B, 10% adenocarcinoma type C, 14% adenoacanthoma b Site of primary tumor not given

Biologic Features

Frequency of Metastases

Only a few reports contain data on the frequency with which spontaneous and induced tumors metastasize to the lung. Table 13 contains data dealing with metastases from spontaneous mammary, liver, and harderian gland tumors. The early reports (Borrell 1903; Bashford and Murray 1904; Murray 1908; Haaland 1911; Marsh 1927, 1929; Pybus and Miller 1934; Ashburn 1937; Dunn 1945, 1953) on pulmonary metastases of spontaneous mammary cancers dealt with percentages and histological appearance. Ashburn (1937) contrasted various percentages of metastasis by whether or not they were detected grossly or histologically in order to eliminate nonmetastatic lesions, while the studies of Consolandi and associates (1958) established the incidence of mammary tumor metastases to the lung for inbred strains of mice with high mammary cancer rates. The percentage of induced hepatic tumors that metastasized to the lungs ranged from 2% to 61 %, with most groups falling between 10% and 20% (Turusov et al. 1973 a; Kyriazis et al. 1974; Vesselinovitch et al. 1978; Frith et al. 1981). Table 14 lists these and several other reports of pulmonary metastases from induced hepatic tumors. Turusov et al. (1973 a) found a low incidence of metastasis from hepatomas and from hepatoblastomas in mice treated with DDT. Frith and associates (1981) also reported metastatic lesions from 14 types of neoplasms in carcinogen-treated mice (Table 14). A low incidence of pulmonary metastasis from mammary and other sites was reported by Turusov et al. (1973 a). Metastasis has been reported for various transplanted tumors, either as "spontaneous" spread from local transplants (Dunham and Stewart 1953; Stewart et al. 1959; Fisher und Fisher 1967; Liebelt and Liebelt 1967; Liebelt et al. 1968) or as from "experimental" systems, such as intravenous inoculation of tumor cells (Fidler 1975; Miller and Heppner 1979; Fidler and White 1981). Transplantable tumors which produced pulmonary metastases included spontaneous or induced tumors in several different inbred strains of mice (Dunham and Stewart 1953). The primary tumors were fibrosarcomas, sarcomas (not otherwise specified), osteogenic sarcomas, rhabdomyosarcomas, melanomas and reticulum cell tumors, and epithelial tumors of the glandular stomach, salivary gland, ovary, testis, cervix, mammary


gland, and skin. The tumors did not often metastasize and occurred late in life. Liebelt and Liebelt (1967) developed a model system for lung metastasis utilizing a transplantable melanoma of (BALBI c x DBAl2f)F1 hybrid mice which, when transplanted subcutaneously, metastasized as early as the second transplant generation; successive transplantation of melanotic or amelanotic tumor lines yielded metastases of the same histologic type as the parent tumor. The frequency of metastasis of mammary tumors obtained from eight mice of two inbred strains and three types of F1 hybrids when transplanted serially by the subcutaneous route for up to 144 generations ranged from 0 to 55% (Liebelt et al. 1968). Under the following headings, we discuss examples of specific host-tumor interrelationships and other factors that have been associated with the occurrence of metastasis.

Tumor Cell Heterogeneity: Tumor cell heterogeneity is a term used to refer to differences in morphology, immunogenicity, growth rate, metabolism, hormone receptors, pigment production, radiosensitivity, and susceptibility to cytotoxic drugs (Fidler 1978; Fidler et al. 1978), Fidler and Kripke (1977), using clones of the B16 melanoma, showed that cells derived from these clones, when injected intravenously, differed dramatically in their ability to establish tumors in the lung. They obtained similar results when cells from an ultraviolet-induced fibrosarcoma cloned in the same manner were injected intravenously. Grdina et al. (1978), who also used fibrosarcoma cells grown in culture and injected intravenously, demonstrated that the intrinsic clonogenic ability of these cells is independent of cell size and age. More important is the ability of a malignant cell to relocate to a site distant from the primary cancer and there be retained and allowed to proliferate (Grdina et al. 1978). The results of Fogel et al. (1979) indicated that cells of tumors with a high metastatic capacity and distinct antigenic properties exist within the tumor cell popUlation and that immunoselection might be involved in the occurrence of lung metastases. Other investigators (Heppner et al. 1978; Nicolson et al. 1978; Talmadge et al. 1979) have since demonstrated heterogeneity for several rodent tumor cell lines. Thus, it appears that murine neoplasms of both long and short origins are heterogeneous, suggesting the presence of several subpopulations within a given cell line. More recently, Fidler and Kripke (1980) cloned sub lines of uniform highly metastatic cells from


single lung metastases. This demonstrated that metastases consist of a more homogeneous population of cells with high metastatic capability than the cells of the primary transplant line.

Genetic Factors (Strain). The frequency of metastasis in mammary-tumor-bearing mice of several inbred strains varies depending on the strain (Consolandi et al. 1958; Liebelt et al. 1968).

Histologic Appearance of the Primary Tumor. Ashburn (1937) divided the primary mammary tumors of 185 mice into six histologic groups. The percentages of metastasis to the lung for each histologic type were as follows: 51.6% cystadenocarcinoma, 41.2% adenocarcinoma, 24.2% papillary cystadenocarcinoma, 10.0% "adenoma malignum," and no cystadenoma, suggesting that the cystadenocarcinomas are clinically more malignant than the tumors in the other groups. The studies of Consolandi et al. (1958) disclosed different percentages of pulmonary metastases depending on the histologic pattern of the primary mammary tumor: type B had 75%, type A 58%, and mixed types A and B had 62%. Apolant (1906), cited by Dunn (1953), pointed out that one could not determine from the histologic examination of a mammary tumor whether it was likely to metastasize to the lungs. More recently, however, van der Valk (1981) reported that 16% of spontaneous mammary tumors of BALB/cfC3H mice metastasize and that such tumors appear less well differentiated than the spontaneous mammary tumors in BALBI c mice. In animals surving to the age of 81-90 weeks, 51 % of the hepatocellular carcinomas with a trabecular pattern metastasized to the lung (Vesselinovitch et al. 1978), irrespective of the chemical carcinogen that induced them. By contrast, only 1.3% of tumors with an adenomatous pattern metastasized, while lesions designated by the authors as hyperplastic nodules of clear, eosinophilic, or basophilic types did not.

Type of Murine Mammary Tumor Virus. The type of murine mammary tumor virus was reported to influence the frequency of metastasis (Squartini and Bistocchi 1977).

Number of Primary Tumors. Metastasis was more frequent in mice having multiple primary mammary cancers than in mice with single tumors (Ashburn 1937); for mice with five to eight tumors, the rate of metastasis was 70%, for mice with four tumors 58.8%, for mice with two tumors

46.6%, and for mice with a single tumor 37.3%. Consolandi et al. (1958) reported frequency of metastasis to be 83% and 60% respectively in mice with multiple tumors and in mice with a single tumor.

Size/Weight of Primary Tumor. Ashburn (1937) reported a correlation between the frequency of metastasis and the size of the spontaneous primary mammary tumor, expressed as the square root of the product of two dimensions in square millimeters. Tumors of 10 mm2 or less, according to the above formula, had an incidence of 6.4%, tumors of 10.1-15 mm2 an incidence of 32.5%, and tumors of30.1-35 mm2 an incidence of69%. Anderson et al. (1974) demonstrated that a large increase in size and weight of the primary mammary tumor rather than its period of growth correlated with the frequency of metastasis. They found that tumors weighing 0.1-0.9 g metastasized at a rate of 2%, those of 0.9-2.0 g at 23%, and those of 2-4 g at 38%. Large tumors weighing 4.1-17 g metastasized at a rate of73%. Several other investigators have reported a direct correlation between the incidence of metastasis and the size of transplantable mouse mammary tumors (Coman 1953; Wood et al. 1954; Wexler et al. 1965; Sugarbaker and Cohen 1972).

Duration of Primary Tumor. Although it is difficult to ascertain accurately the date of tumor appearance, investigators have established their own criteria for establishing the earliest reliable measurement. Ashburn (1937) found that the incidence of metastasis increased with duration of tumor; frequency of metastasis ranged from 20.7% at 11-20 days to 76.5% when over 100 days.

Surgical Ablation. Liebelt and associates (1968) studied the occurrence of pulmonary metastases of spontaneous mammary tumors in strain A mice 60 days after finding a 1.0 x 1.0 em tumor. The incidence in untreated tumor-bearing mice was 39%, in sham-operated mice it was 50%, in mice in which the primary tumor was surgically removed it was 80%, and in mice with surgical removal and ovariectomy it was 42%.

Location of Primary Tumor. Attempts to correlate anatomic site with frequency of metastasis of mammary tumors gave negative results (Williams et al. 1935; Ashburn 1937; Liebelt et al. 1968). On the other hand, Consolandi et al. (1958) found metastatic frequencies of 71 % and 63% for mammary tumors located in the cervical and thoracic

glands respectively, and 62% and 55% for those located in the inguinal and abdominal glands respectively.

Growth Rate and Passage. Ashburn (1937) studied the growth rate of single tumors in mice. The median growth rate of tumors that metastasized was well above that of those that had not. There was an exception, however, with tumors that had been present more than 70 days. Growth rate and metastatic frequency were correlated in the experiments of Consolandi et al. (1958). Heppner and Miller (1981), using transplanted mammary tumor cell lines throughout ten transplant generations, were unable to correlate metastasis and biologic characteristics such as growth and latent period.

Hormonal Factors. Breeding influenced the frequency of metastases of mammary tumors in two strains; that is, breeders had more metastases than virgins (Consolandi et al. 1958; Liebelt et al. 1968; Liebelt et al. 1981) in spite of the fact that virgins -who lived longer developed tumors later than breeders - (Liebelt et al. 1968; Liebelt et al. 1981). Overall breeders developed more tumors (Liebelt et al. 1968). Estrogens and pituitary isografts increased the incidence of metastasis. Other hormones, such as growth hormone, adrenocorticotrophic hormone, and adrenal steroids, promote metastases (Fidler 1975, 1976). Fidler summarized several experiments in which cortisone promoted the growth of tumor cells in the lungs of mice with transplantable mammary cancer, other mouse tumor systems, and intravenously injected melanoma cells. One explanation proposed is that glucocorticoids may alter the capillary endothelial surface, which may lead to increased stickiness and consequent arrest of tumor emboli (Fidler 1976).

Drugs. Lung metastases were enhanced by the cytostatic drugs methotrexate, cytoxan, and 5-fluorouracil (Heppner et al. 1978).

Cell Surface Properties. It is now accepted that changes in the surface properties of tumor cells are important in determining aspects of growth, invasion, and metastasis (Poste and Weiss 1976). Alterations in the surface properties of the cells of the primary tumor contribute to their escape from many restrictions, for instance growth-regulating factors to which normal cells are subject. These factors could include hormones or serum factors that exert their effects by binding to the cell sur-


face. Recently, differences were found in lectinbinding properties between pairs of high and low metastatic murine tumor lines (Dennis et al. 1981; Kerbel et al. 1982; Altevogt et al. 1983). Altevogt et al. (1983) suggested that one could theoretically envision at least three steps in the metastatic cascade that involve cell surface carbohydrate interactions: (a) the release of tumor cells from the primary tumor mass due to altered homotypic adhesion phenomena, (b) the mechanism of blood transportation of metastatic tumor cells by heterotypic cell interactions, and (c) the arrest in organs by specific interactions with the target tissue. Genotypic and phenotypic evolution during progression in vivo toward metastasis was demonstrated using a multiple-drug-marked benign murine tumor cell line by Lagarde et al. (1983).

Motility. In general, increased or excessive cell motility in vivo is a characteristic of malignant cells (Fidler 1974), although more studies have been carried out in experimental systems in vitro than in vivo. The studies of Wood et al. (1954) suggested that the locomotion of tumor cells in vivo is nondirectional and lacks chemotaxis, although other investigators, including Ozaki et al. (1971), suggested that tumor cells secrete a chemotactic factor which may influence motility and invasiveness. Increased motility in vivo is not unique to tumor cells; it is also a characteristic of normal cells during embryogenesis, regeneration, and wound healing (Fidler 1975). Taptiklis (1968, 1969) intravenously injected dissociated cells from cancerous, hyperplastic, and normal thyroid glands. The cells penetrated the endothelial cells of vessels and migrated to extravascular tissues. These cells grew in syngeneic thyroxine-deficient mice but not in normal mice. The normal cells survived as long as 1 year and were even induced to become hyperplastic, "a finding that may help to shed light on the phenomenon of prolonged dormancy and delayed growth of metastatic tumors in man" (Willis 1973).

Irradiation. X-irradiation of host animals before intravenous injection of tumor cells had been reported to increase the incidence of tumor cell implantation in the lung (Fidler 1976). Some investigators attribute this to host immunosuppression, while others suggest that injury to endothelium of pulmonary vessels leads to increase in trapping of tumor cell emboli. However, Suzuki (1983) found that, while preirradiation of murine hosts enhanced growth of tumor cells in the lung follow-


ing intravenous injection, it suppressed lung metastasis from tumors transplanted into the muscles of the leg. These findings strongly suggest that experimental and spontaneous systems of metastasis do not give the same results. Fisher and Kripke (1977, 1978) and Spellman et al. (1977) showed that pulmonary metastases from ultraviolet-induced tumors developed more readily in ultraviolet-irradiated recipients than in nonirradiated recipients. This enhancement was related to a cellular factor, since it could be transferred by lymphoid cells and partially purified Tlymphocytes from the tumor-bearing animals to normal recipients.

Immune Factors. The immunogenicity of the tumor and the immunosurveillance of the host have been implicated in the spread of tumor cells. Ketcham et al. (1966) suggested that the in vivo growth and spread of malignant cells appears to be controlled by some defense mechanism of the host. Crile (1969) proposed that uninvolved reactive or hyperplastic regional lymph nodes in human patients with breast cancer may aid in preventing metastasis if the tumor is small. Vaage and Pepin (1983) concluded that an immunogenic tumor, such as C3H/He mammary cancer, may act as a source of antigen, which can attract a significant number of lymphocytes to function as a temporary accessory lymphoid organ, constituting an early and potent source of systemic immune protective factors. This is manifest as resistance against vascular dissemination and growth of cancer cells. Sugarbaker and Cohen (1972) and Fogel et al. (1979) demonstrated that primary and metastatic tumors vary antigenically from one another. According to Fidler (1980), there have been numerous conflicting reports on the effects of spontaneous or induced tumors in relation to the role of the immune response. Fidler and Kripke (1980) showed that weakly immunogenic tumors metastasized more readily in immunocompetent animals than in immunodeficient animals. The growth and metastasis of a highly antigenic tumor was eliminated by immunologic means in an immunocompetent host. Thus the relationship between immunocompetence and metastasis is far from clear at the present time. Fisher and Kripke (1977, 1978) have demonstrated increased tumor growth in vivo related to an excess of regulatory T cells that suppress antitumor immune response. Fidler (1980) postulated that the factors determining whether inhibition or stimulation of the immune response will predomi-

nate are not known, but probably involve such variables as characteristics of the tumor antigen, the mode of antigen presentation (soluble or cellbound), and the initial site of interaction with host immune cells. Hanna and Schneider (1983) concluded that in mice treated with 17-beta-estradiol there was an association between selective inhibition of natural killer-cell-mediated cytotoxicity in vitro and the enhancement of tumor metastasis. They utilized several different mouse tumor cell lines and suggested the possible role of natural killer cells in natural host resistance against hematogenous tumor dissemination and speculated on an analogous possibility with tumor cells of human origin. Fidler (1974) found that lymphocytes may stimulate or inhibit tumor growth in experimental metastasis, depending on the ratio of lymphocytes to tumor cells. Tumor cells mixed in low ratio with lymphocytes yielded more lung tumor colonies than did tumor cells without the lymphocytes. When they were mixed in a high ratio of lymphocytes to tumor cells, fewer lung metastases resulted. Since clumps of four to five tumor cells result in more lung tumor colonies than single cells, Fidler (1980) postulates that tumor-lymphocyte embolus formation is enhanced by injecting tumor cell clumps, thus increasing the likehood of arrest and tumor growth in the lung.

Tumor Cell Interactions with Extracellular Matrix Barriers. Specific tumor cell products may be associated with the progression of benign tumor cells to malignancy. In vitro tumor cells elaborate degradative enzymes such as proteases, which allow for movement through connective tissue barriers in both murine tumors (Liotta et al. 1977, 1979; Recklies et al. 1982; Van Lamsweerde et al. 1983; Henry et al. 1983) and human tumors (Poole et al. 1978; Recklies et al. 1980). Tumor cells can interact with the extracellular matrix in at least four ways, as described by Liotta et al. (1983): 1. Attachment to the matrix via specific plasma

membrane receptors, such as the glycoproteins laminin and fibronectin. A tumor cell receptor for laminin was reported for the first time by Rao et al. (1983) on BL6 murine melanoma cells and by Terranova et al. (1983) for human MCF-7 breast cancer cells.

2. Degradation of matrix components associated with invasion by specific hydrolytic enzymes such as collagenolytic enzyme for types IV and V collagen of basement membrane. Immuno-

fluorescence and immunoperoxidase techniques used by Barsky et al. (1983) revealed that most invasive carcinomas in humans lacked immuno-reactivity for type IV collagen and laminin of basement membrane, while benign and in situ lesions had intact basement membranes.

3. Increased production of matrix components by host cells in response to the presence of the tumor.

4. Tumor cell synthesis of matrix components.

Vascular Factors. Disseminated intravascular coagulation is associated with the occurrence of lymphocytic leukemia of man or mice. Small and large vessels may be occluded by thrombi, some of which may contain tumor cells. Widespread deposition of fibrin results from activation of the coagulation system, either by entrance of thromboplastic substances into the blood or by endothelial injury. Intravascular coagulation has a twofold effect on the metastasizing tumor. Firstly, since intravascular coagulation compromises and obstructs blood flow to the small vessels, tumor cell emboli are arrested. Secondly, blood flow to tumor cells already located in the lung parenchyma is limited. The ability of actively growing tumors to elicit growth of new capillaries was first observed by Algire and Chalkley (1945). Angiogenesis often initiates the rapid growth (Folkman 1974a), invasion, and metastasis of tumors (Folkman 1974b). Greenblatt and Shubik (1968) demonstrated in vitro a not yet purified diffusible factor from tumor cells that was mitogenic to vascular endothelial cells and stimulated capillaries to proliferate. Gimbrone and Gullino (1976) demonstrated that 30% of mouse mammary HANs elicited neovascularization in the eye. The neovascularization is a useful marker for mouse mammary hyperplasia, since it appears before malignant transformation can be detected (Brem et al. 1977). Strum (1983) used chick chorioallantoic-membrane-bearing grafts of HANs, plaques, hormonedependent tumors, and hormone-independent tumors from strain GRS to test the ability of these lesions to induce angiogenesis. Fifty percent of the plaques and 63% of the HANs tested were angiogenic. Eighty percent of the hormone-dependent tumors and 97% of the hormone-independent tumors induced angiogenesis. Study of hormone-independent tumors by electron microscopy showed failure of tumor cells to penetrate basal lamina. Pitelka et al. (1980b), cit-


ed in Strum (1983), stated that metastases may be found in lungs of mice with large tumors of the hormone-independent type. Another possible mechanism by which lymphocytes may enhance tumor growth and metastasis is by promoting vascularization. Sidkey and Auerbach (1975, 1976) demonstrated that vascularization in tumor-bearing mice could be induced by lymphocytes either reacting against tumors of operating in a graft vs host reaction. Conclusive tests of effects of metastatic tumors by lymphocyte-induced angiogenesis have not been done. In summary, the attempts to relate specific characteristics with malignancy as measured by the frequency or primary metastasis are not conclusive. Correlations with the histological pattern are dependent, at least in spontaneous mammary cancer, on the samples examined. Current information points to the heterogeneous nature of many neoplasms. Certain tumor characteristics mayor may not be correlated with metastasis, depending on more than one characteristic, for instance, growth rate and duration. Immunologic, hormonal, and possibly other homeostatic mechanisms between the host and the tumors play various roles. Finally, the incidence of pulmonary metastasis appears to vary depending on the tumor system utilized, that is, whether it is an artificial or spontaneous system.


Here we briefly compare examples of metastasis to the lung of tumors in domestic animals and several examples of induced tumors metastatic to the lungs of rats.

Occurrence. The single most important and frequent tumor metastasizing to the lungs of cats and dogs is osteosarcoma. Nielsen et al. (1954) and Owen (1969) report metastatic rates of 45%-60% for the dog, and Brodey et al. (1963) suggested that the postsurgical metastatic rate in dogs approached 100%. Four of 11 feline osteosarcomas metastasized to the lung (Engle and Brodey 1969). By contrast, chondrosarcoma of the sheep (Sullivan 1960) and dog (Brodey et al. 1974) tend to grow more slowly and not metastasize. Osteosarcomas of the rat induced by radioisotopes and chemical carcinogens (dimethylbenzanthracene, 3,4-benzopyrene, and chelated copper compounds) can be used as models for osteosarcomas in dog and man, since they are similar histologically and often metastasize (65% or more)


(Owen 1966, 1967). Metastasis to the lung of canine mixed mammary tomors occurs frequently, but the rate of metastasis has not been accurately determined. Rats treated with estrogens have been reported to have an 8% rate of metastasis of mammary tumors to the lung (Cutts 1966). This rate of metastasis is lower than that reported for mice. Hepatocellular tumors are uncommon in domestic animals; they occur in descending order of frequency in the ox, sheep, dog, cat, pig, and horse. Although four out of 13 liver cell tumors of cattle and two out of 21 liver cell tumors of sheep had metastasized, few metastases were found in the lung (Anderson and Sandison 1968; Anderson et al. 1969). Rats fed hepatocarcinogens can serve as a model of lung metastasis. Richardson and BorsosNachtnebel (1951) induced hepatocellular carcinoma by 3'-methyl-diaminoazobenene; 74% metastasized to the lung.

Ultrastructure. Brooks (1970) observed hyperplastic type B (II) cells in the narrow zone of lung tissue which surrounded mammary cancer metastases in inbred mice (see above). However, in an electron microscopic investigation by Ludatscher et al. (1967) of Morris hepatoma 5123 transplanted into strain Buffalo rats, there was no mention of an alveolar cell response to pulmonary metastasis. Brooks (1970) also cited three ultrastructural studies oftype B hyperplasia directly related to injury of alveolar type A cells in other species. Brooks concludes that the epithelial hyperplasia, in apparent response to the presence of tumors, may depend on the tumor type, the species, and damage to type A cells. Further studies to clarify the nature of this finding will necessitate ultrastructural investigation. The ultrastructural studies of Pitelka et al. (1980b) on metastatic mammary carcinomas in the lungs of mice led to the conclusion that interruptions in the basal lamina of primary and metastatic mammary tumor cells are extremely rare. Such interruptions in basal lamina were also seen in chemically induced mammary tumors of rats (Fisher et al. 1975) and mice (Tarin 1969). These findings suggest that interruptions or discontinuities in the basal lamina of mammary tumors provide pathways for active emigration of malignant cells to the surrounding stroma. Pitelka et al. (1980b) cites several ultrastructural studies of human breast tumors in which there were occasional interruptions in the basal lamina in benign lesions and more

frequent and extensive discontinuities or complete absence of basal lamina in tumors of increasing grades of malignancy. Acknowledgments. We thank Dr. Charles Frith, Intox Laboratories, Little Rock, Arkansas for his contribution of the histological material from which Figs. 184, 185 and 188-195 were obtained, and Mr. Larry Ostby for photomicrography.

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NONNEOPLASTIC LESIONS

Bleomycin-Induced Injury, Mouse: A Model for Pulmonary Fibrosis

Drummond H. Bowden

Gross Appearance

No particular gross features distinguish the developing and established fibrotic lesions induced by bleomycin from other types of injury caused by drugs or other chemicals. Mter a single intravenous dose, fibrosis is multifocal and rarely confluent. Diffuse lobular and lobar fibrosis may be expected following the intratracheal insufflation of the drug.


The sequential events induced by bleomycin are remarkably similar whatever the route of administration. What does vary is the speed of the response; a single intravenous injection of bleomycin induces pulmonary fibrosis in about 2 weeks, whereas twice weekly intraperitoneal injections produce fibrosis only after some 4-8 weeks. Direct administration of bleomycin through the trachea also results in the rapid induction of pulmonary fibrosis. Timing of the morphological events is most precise following a single intravenous injection. The following account describes changes in Swiss albino mice given a single intravenous injection of 120 mg bleomycin per kilogram. The earliest detectable alteration is demonstrated by bronchoalveolar lavage. The number of cells in control animals is usually < 20 x 104, almost all of them macrophages. Between 1 and 3 days after bleomycin, the yield is more than doubled and up to 10% are polymorphonuclear leukocytes. In tissue sections of the lung, perivascular edema and endothelial swelling with vacuolation are observed at 5 days (Fig. 199). These changes are observed earlier in the larger pulmonary vessels than in the microvasculature. In some animals, injury does not progress beyond endothelial swelling with concomitant interstitial edema and egress of inflammatory cells. Such lesions are reversible,

the endothelial cells regenerating with complete restitution of a normal air-blood barrier. The most critical cellular event in the genesis of pulmonary injury is destruction of the thin type I epithelium. Damage to the epithelial barrier, though not directly visible by light microscopy, may be inferred by the exudation of fibrin into the air sacs. If this is massive the animals die; if it is limited or multifocal the animals may survive, but the reparative process usually involves fibrosis. As these changes develop the inflammatory cellular response varies. Initially, cells obtained by lavage and observed in tissue sections are predominantly macrophagic with a variable percentage of granulocytes; later the cellular profile changes, with an increase in lymphocytes and plasma cells, particularly in the perivascular spaces. As fibrosis develops, macrophages trapped in rigid segments of the lung become vacuolated and some binucleate and multinucleate forms are seen (Fig. 200). The evolution of fibrosis is observed in two compartments of the lung, in fibrin-filled alveoli and in the interstitium. Proliferation of fibroblasts is well established between 10 and 14 days and the laying down of collagen is progressive thereafter. Fibrosis, which is predominantly subpleural and most marked in the perivascular and peribronchial spaces, is well established by 2-4 weeks after a single injection of bleomycin. The sequence of reparative events is demonstrated most clearly by autoradiography; endothelial regeneration is rapid and results, usually, in complete structural restoration; epithelial regeneration on the other hand is prolonged and is accompanied by the development of abnormal cellular forms. Inappropriate division of interstitial fibroblasts is associated with the deposition of collagen and the development of a stiff noncompliant lung (Fig. 201). The development of fibrosis following the administration of bleomycin is invariably accompanied by distorted regeneration of alveolar epithelium.

Bleomycin-Induced Injury, Mouse: A Model for Pulmonary Fibrosis 161

Fig. 199 (Above). Lung, mouse. Vascular lesion 7 days after a single intravenous injection of bleomycin. There is endothelial vacuolation, subendothelial edema, and a perivascular cellular exudate, predominantly mononuclear. Hand E, x 500

Early necrosis of type I cells is followed by division of type II cells but, instead of differentiating to reconstitute a thin barrier of type I cells, the proliferated cuboidal cells persist, creating tubule-like alveoli. The cuboidal cells may exhibit a variety of metaplastic changes; giant forms almost filling the lumen are observed, together with

(Fig. 200 (Below). Lung, mouse. Alveolar exudate 7 days after a single intravenous injection of bleomycin. The predominant cell is the macrophage; binucleate and multinucleate forms are not uncommon. Hand E, x 500

ciliated alveolar cells and squamous differentiation with keratin production. Such abnormal epithelial regeneration, though seen following a single intravenous injection of bleomycin, is more pronounced after regular intraperitoneal injections given over a period of some weeks (Fig. 202).

162 Drummond H. Bowden

Fig.201 (Above). Lung, mouse, after twice weekly intraperitoneal injections of bleomycin for 4 weeks. Dense bands of subpleural, interstitial collagen distort the normal architecture, producing a honeycomb pattern. Silver methenamine, x 350 (reduced by 10%)

Fig.202 (Below). Lung, mouse, 12 weeks after twice weekly intraperitoneal injections of bleomycin. No normal alveoli are seen; many of the alveolar epithelial cells bear cilia. H and E, x 1000 (reduced by 10%)

Fig. 203. Lung, mouse. Small pulmonary blood vessel 2 days after a single intravenous injection of bleomycin. There is severe cytoplasmic edema of the endothelial cells. Hand E, x 8000 (reduced by 20%)

Fig. 204. Lung, mouse. Alveolus 10 days after a single intravenous injection of bleomycin. Some of the type I cells (EP1) are intact; others (EP1(N)) show focal necrosis allowing the exudation of fibrin (F) from the capillaries (C) into the alveolar lumen (A). TEM, x 6000 (reduced by 20%)

Fig.205. Lung, mouse. Alveolus (A) 3 weeks after a single intravenous injection of bleomycin. A huge type II (EP2) epithelial cell occupies much of the alveolar lumen. TEM, x 6000 (reduced by 20%)


164 Drummond H. Bowden

Ultrastructure

The earliest lesions observed with the electron microscope, endothelial blebbing and subendothelial edema, are evident within 2 days of the injection of bleomycin (Fig. 203). Necrosis of type I alveolar cells is prominent between 7 and 10 days and this breach of the wall is accompanied by exudation of fibrin into the air sacs (Fig. 204). Type II cells and bronchiolar cells show no evidence of injury. Regenerative activity is prompt, endothelial cells are rapidly replaced, and by 12 days many of the alveoli are lined by cuboidal cells derived from dividing type II cells. Whereas in some alveoli normal differentiation of type II cell to type I cell occurs, in others cuboidal cells persist and a variety of metaplastic forms are observed (Fig. 205). Ultrastructural studies confirm the two components of the fibrotic response. In the early phase fibroblastic activity is directly related to the fibrinous exudate which follows necrosis of alveolar cells. These lesions tend to resolve. Fibrosis within the interstitium is progressive and almost constantly associated with epithelial metaplasia.


The pulmonary cells most susceptible to airborne or blood-borne injury are the type I epithelial cells and the endothelial cells. The response of these cells to injury by agents as diverse as viruses, oxidant gases, and blood-borne drugs is similar, the results being dependent upon the dosage and the duration of the insult. Rapid regeneration of both lining layers is the rule, ensuring restitution of the thin air-blood barrier. Delayed or abnormal regeneration or failure to cover the surface wound is associated frequently with proliferation of interstitial fibroblasts and the development of fibrosis. What distinguishes bleomycin-induced injury from other forms of drug-related pulmonary disease is the unusual epithelial response involving delayed and prolonged regeneration with inappropriate differentiation of the proliferated type II cells. Although similar lesions have been described in mice infected with paramyoxoviruses (see page 193), the pattern of abnormal epithelial regeneration associated with interstitial fibrosis is certainly unusual in other forms of drug-induced injury to the lung. The other pathological features, such as the early endothelial injury with leakage of plasma constituents and in-

flammatory cells followed by rapid regeneration and restitution of vascular integrity, represent the standard reparative response of the lung.

Biologic Features

Bleomycin is an antibiotic derived from Streptomyces verticillatus(Umezawa 1974). As a cytotoxic agent it has been particularly useful in the treatment of squamous cell carcinoma, testicular tumors, and lymphomas. It soon became apparent that bleomycin, introduced largely because it has no major toxic effects on kidney or bone marrow, injures the lung. In all cells, normal or neoplastic, bleomycin is degraded by an enzyme which cleaves carboxyamide groups. The responsiveness of a particular cell type to the drug is related to the activity of this enzyme. Epidermal cells in particular contain low levels of the inactivating enzyme, so that bleomycin is able to reach the nucleus, where it induces fragmentation of DNA with subsequent block or derangement in the synthesis of DNA, RNA, and protein. These effects are most pronounced in cells entering the mitotic cycle.

Pathogenesis. The primary location of lesions in the endothelial cells of larger pulmonary vessels is related to the blood-borne delivery of the drug. This is in contrast to injury induced by oxidant gases, where the cells exposed to maximal concentrations in the capillaries are preferentially injured (Adamson and Bowden 1974). The circulating drug enters the pulmonary endothelium, and the particular sensitivity of these lining cells is probably related to lack of the inactivating enzyme. Endothelial injury facilitates fluid transfer to the interstitium and, if the injury is not severe or widespread, rapid regeneration occurs and the animal recovers. Injury to the type I epithelium appears to be a critical event, since disruption of this cellular barrier permits egress of plasma proteins into the alveoli. Such alveolar exudates may be phagocytosed by macrophages and cleared or they may become organized by fibroblasts. Persistence of intraalveolar fibroblastic nodules is unusual, and it is presumed that they are digested by collagenases and phagocytosed. The response of the type II cell may be the determining factor in the genesis of interstitial fibrosis. In most processes of healing, delay in covering a denuded surface may result in unbridled proliferation of connective tissue cells with productive fibrosis. Bleomycin is rapidly taken up by endothelial cells and by type I and type II epithelial


Table 15. Pulmonary toxicity of bleomycin

Animal

Mouse (Swiss albino)

Hamster Dog Baboon Pheasant

Route of administration

Intraperitoneal

Intravenous

Intratracheal Intravenous Intramuscular Intravenous

a 1 mg is approximately 1 unit of bleomycin sulfate

cells. The attenuated lining cells on both sides of the air-blood barrier are readily injured, whereas the type II cell, although it takes up the drug, is not obviously affected. The natural response of these cells to necrosis of adjacent type I cells is to divide. The dividing cell, replete with bleomycin, is most vulnerable to DNA injury and the abnormal cellular forms observed in the reparative phase may be explained in this way (Adamson and Bowden 1977, 1979). While the precise mechanism for the release of fibroblastic activity is not known, the association between delayed or disturbed epithelial restitution and the occurrence of interstitial fibrosis is established. The cellular response in the lung suggests that an immunologic mechanism may be involved in the genesis of the lesions. Cell-mediated immunity has been proposed as the likely pathway, but the induction of fibrosis in the thymicdeficient nude mouse indicates that an intact cellmediated immune system is not essential to the development of pulmonary fibrosis in the mouse. Reported changes in T-Iymphocytes in patients given bleomycin are likely to be secondary reactions only (Elson et al. 1977).

Frequency of Pulmonary Lesions. In the Swiss albino mouse the frequency and severity of pulmonary lesions are dose dependent. A single intravenous injection of 120 mg bleomycin per kilogram results in a 50% mortality rate and the surviving animals develop pulmonary fibrosis in 2-4 weeks. Twice weekly injections of 20 mg bleomycin per kilogram kill half of the animals and induce fibrosis in the survivors in 4-8 weeks. The frequency of metaplastic epithelial changes is greatest in animals receiving intraperitoneal bleomycin for at least 8 weeks. Variability of response in different strains of mice is well recognized, with some being unusually resistant to the drug. Following the intratracheal ad-

Dose" Reference

20 mg/kg twice weekly Adamson and Bowden (1974, 1979)

120mg/kg Adamson and Bowden (1977, 1979)

5 units/kg Snider et al. (1978) OAmg/kg Fleischman et al. (1971) 1.5 units/kg twice weekly McCullough et al. (1978) 4.12 mg/kg twice weekly Bedrossian et al. (1977)

ministration of bleomycin, collagen production and deposition is high in C57BlI6 mice, intermediate in DBAI2 and Swiss mice, and low in BALB mice (Schrier et al. 1983). It is not known if the variation in response is due to differences in the activity of the degradative enzyme in the alveolar cells in these particular strains. A further possibility is suggested by the studies of Walford and Bergmann (1979), who demonstrated a relationship between the main histocompatibility complex and the ability of cells to repair DNA.


The pulmonary toxicity of bleomycin varies considerably from species to species. In dogs, as little as 0.4 mg/kg induces pulmonary lesions, whereas the oorresponding dose in mice is 20 mg/kg by repeated intraperitoneal injection and 120 mg/kg given as a single intravenous injection (Table 15). In humans, toxic effects were quite frequent when the drug was first introduced. Later, as dosage was reduced, pulmonary complications were much less frequent. The occasional severe reaction to doses as small as 5 mg/kg suggests that individual susceptibility may be an important parameter in determining the response.

References

Adamson IYR, Bowden DH (1974) The pathogenesis of bleomycin-induced pulmonary fibrosis in mice. Am J Pathol77: 185-198

Adamson IYR, Bowden DH (1977) Origin of ciliated alveolar epithelial cells in bleomycin-induced lung injury. Am J Pathol87: 569-580

Adamson IYR, Bowden DH (1979) Bleomycin-induced injury and metaplasia of alveolar type 2 cells. Relationship of cellular responses to drug presence in the lung. Am J Pathol96: 531-544

166 Yohko Emi and Yoichi Konishi

Bedrossian CWM, Greenberg SD, Yawn DH, O'Neal RM (1977) Experimentally induced bleomycin sulfate pulmonary toxicity. Histopathologic and ultrastructural study in the pheasant. Arch Pathol Lab Med 101: 248-254

Elson N, Szapiel S, Fulmer J, Crystal R (1977) Role of cellmediated immunity in bleomycin-induced pulmonary fibrosis. Am Rev Respir Dis 115 (Suppl): 54 (abstract)

Fleischman RW, Baker JR, Thompson GR, Schaeppi UH, Ilievsky VR, Cooney DA, Davis RD (1971) Bleomycininduced interstitial pneumonia in dogs. Thorax 26: 675-682

McCullough B, Schneider S, Greene ND, Johanson WG Jr (1978) Bleomycin-induced lung injury in baboons: alteration of cells and immunoglobulins recoverable by bronchoalveolar lavage. Lung 155: 337-358

Schrier DJ, Kunkel RG, Phan SH (1983) The role of strain variation in murine bleomycin-induced pulmonary fibrosis. Am Rev Respir Dis 127: 63-66

Snider GL, Celli BR, Goldstein RH, O'Brien JJ, Lucey EG (1978) Chronic interstitial pulmonary fibrosis produced in hamsters by endotracheal bleomycin. Am Rev Respir Dis 117: 289-297

Umezawa H (1974) Chemistry and mechanism of action of bleomycin. Fed Proc 33: 2296-2302

Walford RL, Bergmann K (1979) Influence of genes associated with main histocompatibility complex on deoxyribonucleic acid excision repair capacity and bleomycin sensitivity in mouse lymphocytes. Tissue Antigens 14: 336-342

Endogenous Lipid Pneumonia in Female B6C3Fl Mice

Yohko Emi and Yoichi Konishi

Synonyms. Cholesterol pneumonia; foamy cell ularly distributed with no specific localization. pneumonia. The nodules are soft and sharply demarcated

from the slightly red surrounding tissue (Fig. 207).

Gross Appearance

Cutaneous application of methylnaphthalene in acetone to mice (to be described later), is followed by lesions in the lungs (Fig. 206). The pleural surface bears multiple white spots and nodules, irreg-

Fig. 206. Gross appearance of lungs, mouse, following cutaneous application of methylnaphthalene. Note irregular surfaces and multiple white spots and nodules


Two lesions are commonly observed. One lesion is characterized by foamy cells and cholesterol

Fig. 207. Cut surface of the lungs, mouse, after fixation

Fig.20S (Above). Endogenous lipid pneumonia, lung of mouse treated with methylnaphthalene. Foamy cells in the alveoli, cholesterol crystals, giant cell reaction, and type II pneumocyte proliferation. Hand E, x 200 (reduced by 15%)

crystals in the alveoli and multinucleated giant cell reaction (Fig. 208). Alveolar walls are slightly thickened and type II pneumocytes are hypertrophied and increased in number. The second lesion is focal alveolar dilatation adjacent to the first le-

Endogenous Lipid Pneumonia in Female B6C3Fl Mice 167

Fig.209 (Below). Endogenous lipid pneumonia, lung of mouse treated with methyl naphthalene. Note compensatory focal dilatation of alveoli adjacent to lesion filled with foamy cells. Hand E, x 40 (reduced by 15%)

sion. This emphysema is probably compensatory due to the first lesion (Fig. 209). Inflammatory cells are only seen in alveolar walls of the surrounding lung tissue. Foamy cell accumulation is not observed in organs other than the lung.

168 Yohko Emi and Yoichi Konishi


Grossly, endogenous lipid pnellmonia resembles the appearance of adenoma of the lung but is different in color and consistency. The induced nodules seen in the pneumonia are relatively soft and white, not translucent, while adenomas are grayyellow, translucent, and firm. Histolo.gi~ally, l~~id pneumonia is recognized as a ch~omc mtersht.tal pneumonitis with the presence of mtracellular hpid deposits, and can be differentiated from other forms of pneumonia. Lipid pneumonia and pulmonary lipidosis have a very similar histological appearance. . . Pulmonary lipidosis is foamy cell accumulatIOn m the alveolar space without interstitial pneumonitis. In lipid pneumonia, destruction of foam cells often results in the formation of cholesterol crystals accompanied by a giant cell reaction. Exogenous lipid pneumonia has a different histological pattern from endogenous lipid pneumonia. The lesions of exogenous lipid pneumonia are focal, whereas those of endogenous lipid pneumonia are diffuse. Exogenous lipid pneumonia is also characterized by granulomatous changes including flattened macrophages and occasional giant cells.

Biologic Features

Exogenous lipid pneumonia is generally caused by oil entering the trachea and being aspirated. Endogenous lipid pneumonia may occur alone, behind a bronchial obstruction, in association with other inflammatory lesions, or in the absence of apparent cause in the lung. In rats endogenous lipid pneumonia can be induced a~te~ prolon~ed breathing of an atmosphere contammg particulate antimony trioxide or instillation of similar material intratracheally (Gross et al. 1952). In our studies, methylnaphthalene dissolved in acetone was painted on the shaved skin of the back of female B6C3F1 mice at doses of 29.7 or 118.8 mg/kg body weight, twice a week for life. Control mice received acetone in the same manner. Methylnaphthalene is not believed to vaporize. Mice which had died or been killed were autopsied and the principal internal organs and the skin were examined histologically. The lesions observed in the lung were diagnosed as endogenous lipid pneumonia. The frequency of pneumonia observed in the three groups was none out of four (controls), three out of 11 (low dose) (27%), and 31 out of32 (high dose) (97%). It was observed as early as 10 weeks after the start of

the experiment in mice that died. Death peaked at 38 weeks; endogenous lipid pneumonia was regarded as the cause of death. The experiment was terminated at 61 weeks. Spontaneous endogenous lipid pneumonia has not been reported in laboratory animals. The results with methylnaphthalene suggest that endogenous lipid pneumonia could result from systemic administration of a chemical agent, although additional exposure by oral or pulmonary routes was not avoided by this experiment. Genetic factors may also be involved, since familial endogenous lipid pneumonia with or without hypercholesterolemia occurs in man (Kinoshita et al. 1970). Cholesterol crystals seen in endogenous lipid pneumonia may be formed by the destruction of foamy cells. Foamy cells contain lipids, mainly phospholipids, synthesized by type II pneumocytes. Histologically, hypertrophy and proliferation of type II pneumocytes are predominant features in this type of pneumonia. Type II pneumocytes, therefore, play an important role in the development of endogenous lipid pneumonia.

Comparison of Lesions in Humans

Endogenous lipid pneumonia seen in mice treated with methylnaphthalene is severe and causes death in some animals. The histology of the pneumonia in mice is quite similar to that described in humans, except that interstitial fibrosis is not observed in mice but is seen in human (Waddell et al. 1954; Wright and Heard 1976). The relation between lipid deposition and interstitial pneumonitis is not known. Endogenous lipid pneumonia in mice treated with methylnaphthalene does provide a useful model for studying the ~ech~nisms of this disease, and may have future Imphcations for the diagnosis and treatment of the human counterpart.

References

Gross P, Brown JHU, Hatch TF (1952) Experimental endogenous lipid pneumonia. Am J Pathol28: 211-218

Kinoshita Y, Ogima I, Saito H, et al. (1970) A case of essential familial hyperlipemia with marked intracellular lipid storage. Nippon Naika Gakkai Zasshi 59: 971-979 (in Japanese) .

Waddell WR, Sniffen RC, Whytehead LL (1954) The etIology of chronic interstitial pneumonitis associated with lipid deposition: an experimental study. J Thorac Surg 28: 134-144

Wright GP, Heard BE (1976) The lungs. In: Sy~~ers W St C (ed) Systemic pathology, 2nd edn. vol 1. LlVlngstone, Edinburgh, pp 373-374

Pulmonary Lipidosis, Rat 169

Pulmonary Lipidosis, Rat

Yohko Emi, Ryuichi Higashiguchi, and Yoichi Konishi

Synonym. Alveolar histiocytosis.

Gross Appearance

The weight of affected lungs (as a percentage of the body weight) does not differ significantly from lungs of control rats. The lungs are normal in color but contain multiple small (ca. 1-2 mm) white spots.


Multiple focal lesions are present with alveoli filled with foamy cells (Fig.210). Some of these cells are macrophages with abundant, lipid-laden cytoplasm, but others represent proliferation of type II pneumocytes in the alveolar walls (Fig. 211). In a rare large lesion, cholesterol crystals may be present. Interstitial pneumonitis is not observed in the surrounding lung.


Pulmonary lipidosis can be differentiated from lipid pneumonia. Animals generally do not die of pulmonary lipidosis but may succumb to lipid pneumonia. Grossly, the lesions of pulmonary lipidosis are small and sometimes difficult to recognize. Histologically, granulomatous changes observed in exogenous lipid pneumonia are not seen in pulmonary lipidosis. Endogenous lipid pneumonia is a form of chronic interstitial pneumonitis with the presence of foamy macrophages and proliferation of type II pneumocytes; pneumonitis is absent in pulmonary lipidosis. Accumulation of foamy cells is more prominent in endogenous

lipid pneumonia and sometimes results in destruction of alveolar walls. In pulmonary lipidosis, reactive foamy cells accumulate in the alveoli but the alveolar wall is relatively well preserved and giant cell reaction is seldom seen.

Biologic Features

Serum biochemistry values (Table 16) are elevated for total cholesterol, ester cholesterol, and betalipoprotein and decreased for high density lipoprotein-cholesterol in hypophysectomized rats. These abnormal values may be caused by general disturbance of lipid metabolism, resulting in pulmonary lipidosis. Pulmonary lipidosis can be induced by anorectic drugs in laboratory animals (Lullmann-Rauch et al. 1972; Gaton and Wolman 1979); the lesions are reversible when administration of the drug is stopped. Myeloid bodies are commonly seen in foamy cells, suggesting that these cells could have originated from type II pneumocytes. Phospholipid accumulation in foamy cells may be related to increased acid phosphatase activity. Pulmonary lipidosis in hypophysectomized rats suggests that hormonal regulation by the hypophysis plays an important role in the development of this disease.

Comparison of Lesions in Humans

Histologically, the lesions are quite similar in animals and man. Epidemiologic studies in Eastern Europe and Russia have shown that pulmonary lipidosis is observed in people who have taken anorectic drugs (Amor et al. 1971; Homann 1974; Hruban et al. 1973; Mach 1972; Rivier et al. 1972;

Table 16. Serum biochemistry of nonoperated and hypophysectomized rats 55 weeks after the operation

Treatment TC HDL-C EC TL TG PL B-LP NEFA

Nonoperated 89.5± 3.5 13 ±1 20± 1 348.5±35.5 111 ±26 177.5±11.5 648± 2 797.5± 2.5 Hypophysectom- 148.5±13.5 6.5±0.5 137±12 405.5±47.5 91.5±10.5 179.5± 0.5 1105±18 633.0 ± 71.0 ized

TC, Total cholesterol; HDL-C, high-density lipoprotein-cholesterol; EC, ester cholesterol; TL, total lipid; TG, triglyceride; PL, phospholipid; P-LP, beta-lipoprotein; NEFA, nonesterified fatty acid

170 Yohko Emi, Ryuichi Higashiguchi, and Yoichi Konishi

Ugriumova 1976). The anorectic drugs are reported to act on feeding centers located in the lateral hypothalamic area and ventromedial hypothalamic nucleus (Baker 1980). In man, pulmonary hypertension frequently accompanies pulmonary lipidosis. Pulmonary lipidosis in hypohysectomized rats may be induced by their decreased food

Fig.210 (Above). Pulmonary lipidosis, lung ofhypophysectomized rat. Alveoli are filled with foamy cells and adjacent tissue is intact. Hand E, x 40 (reduced by 15%)

intake, resulting in changes in body lipid composition. Properly balanced intake of lipids may prevent the development of pulmonary lipidosis.

Fig. 211 (Below). Pulmonary lipidosis, lung ofhypophysectomized rat. Alveoli are filled with lipid-containing foamy cells (a), type II pneumocytes (b), and rare cholesterol crystals (c). Hand E, x 200 (reduced by 15%)

References

Amor H, Schwingshacki H, Dienst! F (1971) Primary pulmonary hypertension. Presentation of 8 cases probably caused by use of an anorectic drug. Minerva Med 62: 4623-4631

Baker CE Jr (ed) (1980) Physicians' desk reference. Medical Economic Company, Oradell

Gaton E, Wolman M (1979) Histochemical study on the pathogenesis of chlorocyclizine-induced pulmonary lipidosis. Histochemistry 63: 203-207

Homann G (1974) Pulmonary hypertension following use of appetite depressants. Med Klin 69: 211

Hruban Z, Slesers A, Aschenbrenner I (1973) Pulmonary

Alveolar Lipoproteinosis, Rat

W.Weller

Synonyms. Alveolar proteinosis; endogenous lipid pneumonia; multifocal histiocytosis; desquamative pneumonia; desquamative interstitial pneumonia (Brewer et al. 1969; Costello et al. 1975; Gaensler et al. 1966; Gough 1967; Liebow et al. 1965; Shortland et al. 1969; Weller 1976).

~.""

L. '~.

~ " , .. '{ .. ,

." ~ ". "

Alveolar Lipoproteinosis, Rat 171

intraalveolar histiocytosis induced by drugs. Toxicol AppIPharmacoI26:72-85

LuHmann-Rauch R, Reil GH, Rossen E, Seiler KU (1972) The ultrastructure of rat lung changes induced by an anorectic drug (chlorphentermine). Virchows Arch (Cell Pathol) 11: 167-181

Mach J (1972) Unusual increase in incidence of pulmonary hypertension. Vnitr Lek 18: 194-196

Rivier JL, Jaeger M, Reymond C, et al (1972) Primary pulmonary arterial hypertension and appetite depressants. Arch Mal Coeur 65: 787 -796

Ugriumova MO (1976) Anorexic preparations and primary pulmonary hypertension. Ter Arkh 48 (2): 126-131

Gross Appearance

The pleural surface of affected rats is quite typical. In initial stages small, white to grayish, round, elevated foci appear on lung surfaces (Fig. 212). If the involvement is minor, these foci can easily be

I . . ~

Fig.212. Lungs, alveolar lipoproteinosis, 12-month-old male Wi star rats

172 W. Weller

overlooked. If the involvement is more extensive, a tendency toward confluence can be observed. The foci appear to expand and become confluent with greater age. In our test series, two different types of additional changes were observed. In one of these, the lesions were nodular, suggesting tumor (Fig.213). The grayish white foci differed in size and were well separated from each other. In confluent lesions (the second type), single foci were not recognizable (Fig. 214); the affected lung surface was gray and slightly elevated in relief.

Fig.213 (Above). Lung, alveolar lipoproteinosis, confluent nodular lesions, 25-month-old Sprague-Dawley rat

Fig.214 (Below). Lung, 23-month-old Wistar rat. Alveolar lipoproteinosis, diffuse lesions


The usual types of tissue changes in alveolar lipoproteinosis are presented in Figs.215 and 216. Initially, the lesions involve alveoli in focal areas (Fig.215) but may become confluent. The alveoli are filled with a moderately dense granular mass, made up essentially of lipid-containing (foamy) macrophages. Increased amounts of protein as well as lipid are present (Beaver et al. 1963; Corrin and King 1969; Flodh et al. 1974; Gross and deTreville 1968; Heppleston 1967; Heppleston et al. 1970; Heppleston and Young 1972; Yang et al. 1966). At higher magnification (Fig. 215), the alveoli are seen to be filled with a partly eosinophilic, partly basophilic, PAS-positive, granular material. In some areas, deposits of cholesterol crystals may be seen, usually engulfed by macrophages. These cells have dense, usually centrally placed nuclei and abundant foamy cytoplasm (Fig. 216). In the center of larger foci, the macrophages are often absent, but some shadowy single-cell structures may be found. In the periphery of these larger foci, macrophages with foamy cytoplasm may be observed in varying numbers. The adjacent alveolar septa of the lung usually appear to be normal, but in some locations numerous cuboidal alveolar cells (type II pneumocytes) can be identified. In none of the animals are inflammatory infiltrates present in alveolar septa or in peribronchial tissue. Most of these foci in animals exposed to dust are free of dust and, on the whole, distinctly separate from zones of early fibrosis, which do contain dust particles. Cholesterol granulomas with multinucleated giant cells have been described (Beaver et al. 1963; Corrin and King 1969; Gross and de Treville 1968; Pittermann 1973; Pittermann and Deerberg 1974; Weller et al. 1974, 1978; Yang et al. 1966). They are infrequent, however, and have no recognizable relationship to the degree of alveolar lipoproteinosis (Fig. 217).

Enzyme Histochemical Findings

In our studies, histochemical investigations employed the folowing enzymes: hydro lases (acid and alkaline phsophatases, nonspecific esterase) and oxidoreductases (DPNH-/TPNH-diaphorase, alpha-glycerophosphate oxidase, succinic dehydrogenase). In most of the type II pneumocytes, enzyme activity was similar to that demonstrated in the same

cells in histologically normal alveoli. In proliferating type II pneumocytes, increased amounts of hydro lases and oxidoreductases were observed. This indicates elevated metabolic activity at the cellular level and correlates with the electron microscropic findings of increased numbers of osmiophilic lamellar bodies in the cytoplasm. The greatest number of cells (macrophages) in the alveoli contained weak or nonreacting enzymes.

Ultrastructure

The light and electron microscopic findings in alveolar lipoproteinosis have been well described. The findings of the present study are not exceptional. Different opinions exist, however, on the identity of the cells that fill the alveoli. Certain authors (Pittermann 1973), some with reservations (Heppleston et al. 1970; Heppleston and Young 1972), consider the cells in alveoli to be type II pneumocytes. In our study these cells were identified as macrophages. Our interpretation agrees with that of Heppleston et al. (1970). The very large, in general uninuclear, macro phages which fill the alveoli contain relatively regular, large, round inclusions confined by a simple membrane. These inclusions are filled with layers of fine osmiophilic lamellae, often concentrically arranged. The cytoplasm of these cells appears only as a dark, narrow matrix between these lamellar particles. Other organelles have not been identified. The nuclei are primarily eccentrically located, and frequently appear to be indented by the inclusion bodies. The inclusion bodies are often adjacent to the cell membrane (Fig. 218). In addition to these round, intracytoplasmic particles, some cells also contain longitudinal and optically empty hollow spaces from which crystalline, needle-shaped deposits had been released during fixation and embedding. The affected intraalveolar type II pneumocytes maintain their usual structures, although the osmiophilic lamellar bodies in these cells increase in number. Microvilli are present on the surface. Distinct finely granular deposits, in part consisting of fine osmiophilic lamellae, are present free

Fig.215 (Above). Lung, rat, alveolar lipoprotein os is. FocalC> lesion with filled alveoli. Hand E, x 25

Fig.216 (Below). Lung, rat, alveolar lipoproteinosis. Small focus of densely packed macrophages with foamy cytoplasm in alveoli. Methylene blue and basic fuchsin, x 630


174 W. Weller

Fig.217. Lung, rat. Subpleural cholesterol granulomas. H and E, x 25

in the alveolar lumen and sometimes located on the alveolar epithelium. Occasionally, these deposits have a fine vesicular arrangement. The small residual alveolar lumen between pneumocytes and macrophages is in some sections filled with these deposits. In the surrounding regions, focal increase of fibers is evident in the interstitium. Interstitial vessels are not changed.


The histologic features of this disease are quite characteristic and usually provide the basis for precise diagnosis. The alveoli in small or large parts of the lung are filled with large lipid-laden cells. The presence of this lipid gives the cytoplasm a foamy appearance. The lesion is readily distinguished from infectious granulomas by the absence of other inflammatory exudate. In cryptococcosis and pneumocystosis, leukocyte response also may be minimal, but the organisms may be identified with special stains (Gomori's methenamine silver, Gridley's fungus stain, PAS).

Fig.218. Lung, rat, alveolar lipoproteinosis. Osmiophilic lamellar bodies in alveolar macrophages. TEM, x 5000

Biologic Features

The different terms for the same syndrome are based on the differing interpretations of the features observed by various investigators. In involved alveoli, protein-containing material is found, which is believed to originate (Heppleston 1967) from decomposing macrophages. Surfactant and lamellar bodies may also contribute to the protein volume (Heppleston and Young 1972). The lipid volume in altered alveoli is of particular importance (Beaver et al. 1963; Corrin and King 1969; Flodh et al. 1974; Heppleston et al. 1970). The proportion of cholesterol, triglycerides, and especially phospholipids is distinctly increased. Therefore, we suggest this disease should be named alveolar lipoproteinosis rather than alveolar proteinosis. The increased phospholipid proportion in alveolar lipoproteinosis suggests the interesting possibility of a relationship to the lung surfactant system (Corrin and King 1969; Heppleston et al. 1970; Heppleston and Young 1972). This disease may impair or invalidate animal experiments and thus be compared to chronic re-

spiratory disease of conventional rats. On the basis of comparison of lesions in the lungs of both species, alveolar lipoproteinosis in rats may serve as a model for human alveolar lipoproteinosis.

Natural History. These changes in the lungs were first described in germ-free rats (Beaver et al. 1963). Further reports followed relative to its appearance in germ-free (Pittermann and Deerberg 1974) as well as specific-pathogen-free rats (Corrin and King 1969; Pittermann 1973; Weller 1977) and conventional rats (Kuhn et al. 1966; Yang et al. 1966).

Morbidity. The morbidity is quite variable and significantly influenced by the age of the rats (Flodh et al. 1974). The percentage of animals with alveolar lipoproteinosis increases as animal groups become older (Weller et al. 1978). According to Beaver et al. (1963), the percentage of animals with alveolar lipoproteinosis in different groups may vary between 0 and 97%. In published statistics the most frequent postmortem finding was alveolar lipoproteinosis (Pittermann and Deerberg 1974). Another report referred to frequencies as high as 20% (Yang et al. 1966). In our tests, the frequency in untreated control anim~ls was as great as 55%. Postmortem findings in ammals aged 12-15 months from different breede!s revealed a frequency of alveolar lipoproteinoSIS between 40% and 90%. The earliest occurrences of the disease, as reported in three studies, were at 6 (Flodh et al. 1974), 11 (Pittermann and Deerberg 1974), and 15 months of age (Yang et al. 1966). Clear statements have not been made concerning the relationship between morbidity and strain of origin or sex of the rats: reports are even contradictory (Flodh et al. 1974; Weller et al. 1~78; Yang et al. 1966). Although a comparison WIth completely healthy animals could not be made due to the small number of unaffected rats mortality does not seem to be significantly influ~ enced. Organ weigh~s in affected animals may be slightly or .severely mcreased. In normal rats the lung ~eIghs up to 1.5 g; rats with alveolar lipoproteinoSIS have lungs that weigh up to 4 g; and in dust-exposed animals, the lungs may weigh as much as 18 g. In hilar lymph nodes, neither macroscopic nor light microscopic changes are observed and the weight of these lymph nodes is not alter;d.

Etiology. When the alveolar lipoproteinosis syn?rome ~as fi~st ~escribed (Beaver et al. 1963) durl~g an m~esttgatlOn of germ-free rats, a cirrhogemc, protem-deficient diet was thought to be the


cause. Subsequent reports postulated a causal influence of dust inhalation (Corrin and King 1969; Gross and deTreville 1968; Heppleston 1967; Heppleston et al. 1970; Heppleston and Young 1972; Jennings et al. 1965). However, when alveolar lipoproteinosis also appeared in untreated rats kept under controlled conditions (Pittermann 1973; Pittermann and Deerberg 1974; Weller 1977; Weller et al. 1974; Yang et al. 1966), it was concluded that an independent lung disease was present in these rats (Weller et al. 1974, 1978). The pathogenesis of alveolar lipoproteinosis is not yet clarified. A variety of potential etiologies have been studied. According to Gross and deTreville (1968) and Heppleston and Young (1972), a disturbance of the bronchial purification mechanism, an excessive production of alveolar macrophages and a lack or insufficiency of autolytic enzymes can be regarded as the first stage in the pathogenesis. An excessive production of surfactant with an increased proportion of phospholipid, together with hyperreactivity of type II pneumocytes (Corrin and King 1969; Heppleston et al. 1970), has also bee~ proposed. This suggestion is supported by the mcreased occurrence of free lamellar bodies in alveoli as well as phagocytized lamellar bodies in alveolar macrophages, a process which may be compared to a merocrine secretion passed from type II pneumocytes into the alveolar lumen (Hatasa and Nakamura 1965). A pathological surfactant has also been described (Heppleston and Young 1972). Cholesterol, neutral fat, and cell decomposition products are able to deactivate normal surfactant. According to Felts (1965), glucose availability is important to li~ometabolism in the lung. Furthermore, due to an lllcreased capillary permeability the contents of alveoli have been regarded as transudates (Heppleston et al. 1970). In addition, an immunologic disturbance has been suggested (Gray 1973). The implications of the cholesterol granulomas (Beaver et al. 1963; Corrin and King 1969; Pittermann 1973; Pittermann and Deerberg 1974; Weller et al. 1974; Yang et al. 1966) need to be clarified. They are probably related to the increased cholesterol concentration in the abnormal lungs. However, only a small proportion of lungs with alveolar lipoproteinosis also contain cholesterol granulomas. According to Kluge (1959), cholesterol supplied intratracheally cannot be regarded as a cause of progressive cholesterol pneumonia. Flodh et al. (1974), however, found an increased number of foamy cells in lungs of rats that had been fed diets rich in cholesterol or triglyceride.

176 W. Weller


Alveolar lipoproteinosis of rats is not only of importance in experimental animals but may also serve as a model for the human disease. The term "pulmonary alveolar proteinosis" was introducted by Rosen et al. (1958) for a human disease that had been previously unknown or misinterpreted. This disease is characterized by a recurring and/ or progressive accumulation of phospholipidcontaining material in the alveoli, but without inflammation. Patients suffer from an increasing dyspnea combined with cough and loss of weight; nearly one-third of them eventually die of cardiorespiratory insufficiency following a protracted illness. Since the first description, many other cases have been reported (Weller et al. 1978). With respect to the comparability of lung changes found in men and in rats, the following can be said: spontaneous alveolar lipoproteinosis in rats and alveolar lipoprotein os is in animals after dust inhalation do not differ significantly by light or electron microscopy or by enzyme histochemistry from that described for human patients. Alveolar lipoproteinosis occurs not only in rats and man, but also in hamsters and guinea pigs (Gross and deTreville 1968).

References

Beaver DL, Ashburn LL, McDaniel EG, Brown ND (1963) Lipid deposits in the lungs of genn-free animals. Arch Pathol 76: 565-570

Brewer DB, Heath D, Asquith P (1969) Electron microscopy of desquamative interstitial pneumonia. J Pathol 97:317-323

Corrin B, King E (1969) Experimental endogenous lipid pneumonia and silicosis. J Pathol97: 325-330

Costello JF, Moriarty DC, Branthwaite MA, Turner-Warwick M, Corrin B (1975) Diagnosis and management of alveolar proteinosis: the role of electron microscopy. Thorax 30: 121-132

Felts JM (1965) Carbohydrate and lipid metabolism of lung tissue in vitro. Med Thorac 22: 89-99

Flodh H, Magnusson G, Magnusson 0 (1974) Pulmonary foam cells in rats of different age. Z Versuchstierkd 16: 299-312

Gaensler EA, Goff AM, Prowse CM (1966) Desquamative interstitial pneumonia. N Engl J Med 274: 113-128

Gough J (1967) Silicosis and alveolar proteinosis. Br Med J 1: 629

Gray ES (1973) Autoimmunity in childhood pulmonary alveolar proteinosis. Br Med J 4: 296-297 (letter)

Gross P, deTreville RTP (1968) Alveolar proteinosis. Its experimental production in rodents. Arch Pathol 86: 255-261

Hatasa K, Nakamura T (1965) Electron microscopic observations of lung alveolar epithelial cells of nonnal young mice with special reference to fonnation and secretion of osmiophilic lamellar bodies. Z Zellforsch 68: 266-277

Heppleston AG (1967) Atypical reaction to inhaled silica. Nature 213: 199

Heppleston AG, Young AE (1972) Alveolar lipo-proteinosis: an ultrastructural comparison of the experimental and human fonns. J Patholl07: 107-117

Heppleston AG, Wright NA, Stewart JA (1970) Experimental alveolar lipo-proteinosis following the inhaltation of silica. J Patholl0l: 293-307

Jennings IW, Gresham GA, Howard AN (1965) Pulmonary lipidosis in laboratory rats. J Reticuloendothel Soc 2: 130-140

Kluge A (1959) Versuche zur Cholesterinpneumonie. In: Verh. Dtsch. Ges. Path. Fischer, Stuttgart, p 274

Kuhn C, Gyorkey F, Levine BE, Ramirez-Rivera J (1966) Pulmonary alveolar proteinosis. A study using enzyme histochemistry, electron microscopy, and surface tension measurement. Lab Invest 15: 492-509

Liebow AA, Steer A, Billingsley JG (1965) Desquamative interstitial pneumonia. Am J Med 39: 369-404

Pittennann W (1973) Pulmonale Alveolar-Proteinose bei alten SPF HAN: Sprague-Dawley-Ratten. XI. Scientific meeting of the Society for Laboratory Animal Science 9-12 May, Antwerp

Pittennann W, Deerberg F (1974) Erkrankungen bei keimfreien Ratten. Fortbildungslehrgang fUr Versuchstierkunde, 8-12 October 1973, Berlin. Institut fUr Versuchstierkunde und Versuchstierkrankheit, University of Berlin

Rosen SH, Castleman B, Liebow AA (1958) Pulmonary alveolar proteinosis. N Engl J Med 258: 1123-1142

Shortland JR, Darke CS, Crane WA (1969) Electron microscopy of desquamative interstitial pneumonia. Thorax 24: 192-208

Weller W (1976) Grundlagen der tierexperimentellen Pneumokonioseforschung. In: WT Ulmer, G Reichel (eds) Pneumokoniosen. Springer, Berlin Heidelberg New York, p 49 (Handbuch der inneren Medizin, vol 4/1)

Weller W (1977) Anthrakosilikose. Tierexperimentelle Forschung. Bergbau-Berufsgenossenschaft, Bochum

Weller W, Haacks H, Guzy J1(, Heine W (1974) Silikoseentwicklung im Intraperitonealtest bei Verwendung von keimfreien, SPF- und konventionellen Ratten. Beitr Silikoseforsch 26: 265-276

Weller W, Kissler W, Morgenroth K (1978) Alveolar-Lipoprotei nose bei Ratten. Z Versuchstierkd 20: 1-18

Yang YH, Yang CY, Grice HC (1966) Multifocal histiocytosis in the lungs of rats. J Pathol Bacteriol 92: 559-561

Bronchiolar/ Alveolar Hyperplasia, Lung, Rat 177

Bronchiolar/ Alveolar Hyperplasia, Lung, Rat

Gary A. Boorman

Synonyms. Alveolar hyperplasia; bronchiolar metaplasia; bronchioalveolar adenomatosis; pulmonary adenomatosis; alveolar epitheliolization.

Gross Appearance

The lesion is generally not visible grossly but may appear as white pinpoint foci from the pleural surface.


In microscopic sections, foci of hyperplasia appear as poorly circumscribed areas of increased cellularity in the lung (Fig. 219). The increased cellularity is due to increased numbers of cuboidal cells lining the alveoli and often intraluminal cells which are predominantly macrophages (Fig. 220). Important features are the lack of compression at the margin (Fig.219) and the persistence of normal alveolar architecture within the lesion (Fig. 220). At the margin the hyperplastic cuboidal epithelium extends along the alveolar surface of contiguous alveoli (Fig. 221). As the lesions progress the lining cells may become multilayered or form papillary projections into the lu-

mina. The cuboidal cells comprising the hyperplastic alveolar epithelium usually resemble normal type II pneumocytes with little atypia and few mitotic figures.


Bronchiolar/alveolar hyperplasia can usually be distinguished from bronchiolar/alveolar adenoma. However, with large foci of hyperplasia the distinction becomes more difficult, since morphologically there appears to be a gradual progression from focal hyperplasia to adenoma and finally to obvious carcinoma with distant metastases. This has created some controversy regarding the appropriate diagnostic criteria to separate these entities. Some of the diagnostic difficulty is due to the lack of significant cytological difference between hyperplasia and bronchiolar/alveolar adenoma. Thus one has to rely on structural features and growth patterns to distinguish the two lesions. In contrast to focal hyperplasia, adenomas have a more complex and/or solid pattern in which the original architecture is difficult to discern. The margin of the lesion is also useful. In hyperplasia, the margin is not well defined. Contiguous alveoli are partially covered by cuboidal (hyperplastic)

Fig.219. Focal bronchiolar/alveolar hyperplasia, lung, rat. The pleural surface is not raised, adjacent lung is not compressed, and the underlying alveolar pattern can be seen throughout the lesion. Hand E, x 120

178 Gary A. Boonnan

Fig.220 (Left). Bronchiolar/alveolar hyperplasia, lung, rat. Alveolar surfaces are lined by a continuous layer of plump cuboidal cells. Much of the increased cellularity is due to free intraluminal cells, which appear to be pulmonary macrophages. Hand E, x 200

cells and there is no evidence of compression. With bronchiolar/alveolar adenoma, the margin between normal and affected tissue is often quite sharp, with compression of adjacent pulmonary tissue. Inflammatory cells such as intraluminal macrophages and perivascular accumulations of lymphocytes are more commonly associated with areas of hyperplasia.

Biologic Features

Several cell types may contribute to the development of focal bronchiolar/alveolar hyperplasia in the distal lung of the rat. The alveolar epithelium is comprised of two cell types, the type I and type II pneumocyte. The main lining cell of the alveolus is the type I pneumocyte, which is very

Fig.221 (Right). Bronchiolar/alveolar hyperplasia, lung, rat. At the margin of the lesion the hyperplastic cells extend partially into contiguous alveoli. No compression or distortion of adjacent alveoli; hyperplastic cells lining the alveoli are unifonn with little cellular atypia, are more basophilic, and contain few mitotic figures. The intraluminal erythrocytes are not a usual feature and their presence is felt to be an agonal event. Hand E, x 400

flattened and covers about 90%-95% of the alveolar surface (Pinkerton et al. 1982). The remaining cells are type II pneumocytes. The type II cell is cuboidal and its cytoplasm contains osmiophilic lamellar inclusions which are believed to be the source oflung surfactant (Kauffman 1980). Injury to the alveolar epithelium resulting in necrosis of type I cells stimulates the proliferation of type II cells to replace the damaged epithelium (Bocking et al. 1981; Mason et al. 1977; Evans et al. 1975, 1976). Hyperplasia of type II cells is, therefore, a normal response contributing to the repair of the epithelial surface. Cells of the terminal bronchiole also may proliferate and migrate into alveoli in response to lung injury (Aso et al. 1976). Cells of the intrapulmonary airways that have regenerative capability and possibly contribute to foci of hyperplasia include

Table 17. Bronchiolar/alveolar hyperplasia in F344 rats

Study typea

Gavage Other

Total

Males

7/249 (2.8%) 4/198 (2.0%)

11/447 (2.5%)

Females

4/248 (1.6%) 31200 (1.5%)

7/448 (1.6%)

a Incidence rates from control groups. Animals in gavage studies received com oil 5 mllkg, five times/week for 104 weeks. Includes data from nine NTP technical reports issued through 1983

nonciliated bronchiolar (Clara) cells, Kulchitsky (APUD) cells, and ciliated cells (Jeffery and Reid 1975). Bleomycin exposure causes the appearance of ciliated cells in alveoli (Kauffman 1980), ozone exposure causes focal clusters of nonciliated bronchiolar (Clara) cells (Boorman et al. 1980), and nitrosamine will increase Clara and APUD cells (Kauffman et al. 1979). Thus a variety of cell types have the potential to proliferate and to be found in areas of hyperplasia. The type II pneumocytes have a higher mitotic potential (Dormans 1983) and are felt to be the major cell type found in areas of hyperplasia. It must be cautioned, however, that the role of other cell types has not been thoroughly established. Increased emphasis on toxic lesions in carcinogenesis studies has resulted in better documentation of hyperplastic lesions (Ottolenghi et al. 1975; Pour et al. 1976). In nine recent studies reported by the National Toxicology Program, the incidence of bronchiolar/alveolar hyperplasia is quite common in control groups of rats (Table 17). In a recently completed inhalation study of chrysotile asbestos fibers, bronchiolar/alveolar hyperplasia was a frequent finding and it may represent an early change in the spectrum of lesions leading to bronchiolar/alveolar carcinoma (McConnell, in preparation). Studies relating specific morphological features to biologic behavior, such as progressive growth following transplantation, remain to be done.

Bronchiolar/ Alveolar Hyperplasia, Lung, Rat 179

References

Aso Y, Yoneda K Kikkawa Y (1976) Morphologic and biochemical study of pulmonary changes induced by bleomycin in mice. Lab Invest 35: 558-568

Bocking A, Mittermayer C, von Deimling a (1981) Urethane-induced lung hyperplasia. Lab Invest 44: 138-143

Boorman GA, Schwartz LW, Dungworth DL (1980) Pulmonary effects of prolonged ozone insult in rats. Lab Invest 43: 108-115

Dormans JA (1983) The ultrastructure of various cell types in the lung of the rat: a survey. Exp Pathol24: 15-33

Evans MJ, Cabral D, Stephens RJ, Freeman G (1975) Transformation of alveolar type II cells to type I cells following exposure to N02. Exp Mol Pathol 22: 142-150

Evans MJ, Johnson LV, Stephens RJ, Freeman G (1976) Cell renewal in the lungs of rats exposed to low levels of ozone. Exp Mol Pathol24: 70-83

Jeffery PK Reid L (1975) New observations of rat airway epithelium: a quantitative and electron microscopic study. J Anat 120: 295-320

Kauffman SL (1980) Cell proliferation in the mammalian lung. Int Rev Exp Pathol22: 131-191

Kauffman SL, Alexander L, Sass L (1979) Histologic and ultrastructural features of the Clara cell adenoma of the mouse lung. Lab Invest 40: 708-716

Mason RJ, Dobbs LG, GreenleafRD, Williams MC (1977) Alveolar type II cells. Fed Proc 36: 2697 -2702

National Toxicology Program (NTP) (1983) Technical report series, National Technical Information Service, US Department of Commerce, Springfield

Ottolenghi AD, Haseman JK Payne WW, Falk HL, MacFarland HN (1975) Inhalation studies of nickel sulfide in pulmonary carcinogenesis of rats. JNCI 54: 1165-1172

Pinkerton KE, Barry BE, O'Neil 11, Raub JA, Pratt PC, Crapo JD (1982) Morphologic changes in the lung during the lifespan of Fischer 344 rats. Am J Anat 164: 155-174

Pour P, Stanton MF, Kuschner M, Laskin S, Shabad LM (1976) Tumours of the respiratory tract. In: Turusov VS (ed) Pathology of tumours in laboratory animals, vol 1. Tumours of the rat, part 2. IARC, Lyon, pp 1-40 (Publication no 6)

180 G.E.Dagle and A.P. Wehner

Fly Ash Pneumoconiosis, Hamster

G. E. Dagle and A. P. Wehner

Synonyms. Pneumoconiosis is the generic term for the presence of different dusts in the lung and the resultant lesions. Anthracosis, the condition related to soot in the lungs, may include fly ash and/or other combustion products of fossil fuels (Sanders et al. 1980).

. -

A B

Gross Appearance

The lungs are diffusely tan to gray, with black foci, typically 1-2 mm in diameter, that tend to be distributed in subpleural areas (Fig. 222). Lung weights and volumes are increased after prolonged exposure .

Fig. 222 A, B. Gross appearance oflungs from hamsters after 16 months of exposure to A low or B high concentrations of fly ash aerosol

Fig. 223. Interstitial reaction in lung of hamster exposed to fly ash. Hand E, x 486 (reduced by 10%)


Dust, generally phagocytosed by alveolar macrophages, aggregates in alveoli throughout all lobes, with a predilection for peribronchiolar and subpleural areas (Fig. 223). An interstitial reaction, composed of thickened alveolar septa with prominent alveolar epithelial cells and collagenous stroma, is associated with the dust accumulation (Fig. 224). Bronchiolization (see "Bronchiolar/

Fig.224 (Above). Bronchiolization (hyperplasia) (straight arrows) and interstitial reaction (curved arrow) in lung of hamster exposed to fly ash. Hand E, x 120 (reduced by 10%)

Fly Ash Pneumoconiosis, Hamster 181

Alveolar Hyperplasia" p 175), composed of proliferation of bronchiolar epithelium into alveolar ducts, is more pronounced in areas of peribronchiolar dust accumulation (Fig. 225). Vesicular emphysema occasionally occurs.

Ultrastructure

The ultrastructural features of this lesion in hamsters has not been described.

Fig.225 (Below). Bronchiolization (hyperplasia) in lung of hamster exposed to fly ash. Hand E, x 486 (reduced by 10%)

182 G. E. Dagle and A. P. Wehner


The lesions are associated with relatively high concentrations of dust in the lung. Definitive diagnosis would depend upon X-ray or diffraction patterns from individual dust particles. Ferruginous bodies and multinucleated giant cells, as formed with asbestos, are lacking. The lesions also lack the interstitial fibrosis and alveolar proteinosis associated with silicosis. The epithelial proliferation of alveoli and bronchioles is limited to the immediate area of dust accumulation. Since prolonged exposure to high levels of dust is needed to produce the lesions, dust will always be present in close association with lesions.

Biologic Features

Natural History. The effects of inhaled fly ash were studied in the Syrian golden hamster exposed to 70 ~g/l fly ash for 6 hi day, 5 dayslweek for up to 20 weeks (Wehner et al. 1981). Dust deposition increased with time and decreased with recovery. The interstitial reaction increased with time but did not diminish significantly after exposure stopped. Bronchiolization also increased with duration of treatment and occasionally progressed even after exposures ceased. Mortality for exposed animals was not higher than for controls (Wehner et al. 1981).

Pathogenesis. Dust is phagocytosed by alveolar macrophages; the release of enzymes from alveolar macrophages injures alveolar epithelium. This is followed by compensatory hyperplasia of alve-

olar and brochiolar epithelium and, if injury is severe, fibrosis (Sanders et al. 1980). Direct effects of fly ash minerals or absorbed materials on epithelium cannot, however, be ruled out. Dust is cleared from the lung by mucociliary escalator or accumulates in mediastinal lymph nodes.

Etiology. Fly ash consists of microcrystals and micro spheroids of varying structure, size, and composition - in general, silicates and oxides of aluminum and other minerals that remain after complete combustion of coal. Various materials from the atmosphere may also adhere to the surfaces of the particles. Thus it is rather difficult to attribute to any specific component a given toxicl pathogenic effect observed after exposure to fly ash.

Frequency. Lesions observed in hamsters exposed to aerosols of fly ash for 6 hi day for 20 months are quantified in Table 18.


Anthracosis, including that caused by fly ash, is regarded as an innocuous condition in humans (Spencer 1977). Studies of primates exposed to flay ash, at levels lower than those for hamsters in our study, demonstrated lesions "similar to that seen in any 'nuisance' dust" which were "not considered to be evidence of permanent pathological alteration" (MacFarland et al. 1968). Recently, however, it has been suggested that fly ash particles may act as carriers for toxic materials or may be fibrogenic per se in man (Golden et al. 1982).

Table 18. Histological changes related to fly ash exposure of hamsters for 20 months

Lung change observed Exposure Total no. of No. of hamsters Average severity level hamsters with lesions of lesions' (llg/l3)

Dust accumulation 70 61 61 3.25b

17 62 62 2.29bc

0 55 0

Interstitial reaction 70 61 61 2.23b

17 62 56 1.39bc

0 55 4 1.00

Bronchiolization 70 61 44 1.45b

17 62 29 1.17cd

0 55 11 1.18

a Very slight (or very small amount) = 1, slight (or small amount) = 2, moderate = 3, marked = 4, and severe = 5 b Significantly different from sham-exposed controls, p < 0.01 C Sigificantly different from 70-llg/l exposure level, p < 0.01 d Significantly different from sham-exposed controls, p < 0.05

References

Golden EB, Warnock ML, Hulett LD Jr, Churg AM (1982) Fly ash lung: a new pneumoconiosis? Am Rev Respir Dis 125: 108-112

MacFarland HN, Ulrich CE, Martin A (1968) Chronic exposure of cynomolgus monkeys to fly ash. In: Walton WH (ed) Inhaled particles. Unwin, Surrey

Sanders CL, Cross FT, Dagle GE, Mahaffey JA (eds)

Asbestosis, Hamster

G. E. Dagle and A. P. Wehner

Synonym. Asbestos pneumoconiosis.

Gross Appearance

At the higher exposure levels (23 ~g/l), the lungs have a mottled appearance with slightly rusty-tan areas replacing the normal pink coloration. Lung weights tend to increase after prolonged exposure.


Increased numbers of alveolar macrophages and ferruginous bodies are the principal lesions observed at lower exposure levels. The macrophages are diffusely distributed, with some aggregation in areas around respiratory bronchioles (Fig. 226). Those engulfing the ferruginous bodies are generally aggregated in alveoli and frequently form multinucleated giant cells (Fig.227). The ferruginous bodies measure up to 20 ~m long and 2 ~m thick, are frequently curved, stain golden yellow with hematoxylin and eosin, and have a typical beaded appearance due to the hemosiderin deposited on the asbestos fibers. Numerous macrophages contain two or more ferruginous bodies. Portions of the ferruginous bodies frequently appear incompletely phagocytosed. Only rarely are ferruginous bodies seen outside of phagocytes. A mild interstitial fibrosis occurs after 7 months of exposure (Wehner et al. 1975). This occurs in areas with aggregated alveolar macrophages and, at high exposure levels, is associated with lymphocytes and plasma cells (Fig. 228). Proliferation of alveolar and bronchiolar epithelium and cho-

Asbestosis, Hamster 183

(1980) Pulmonary toxicology of respirable particles. Proceedings: DOE symposium series, CONF-791002. National Technical Information Service, DOE, Oak Ridge

Spencer H (1977) Anthracosis. In: Spencer H (ed) Pathology of the lung, vol 1. Saunders, Philadelphia p411

Wehner AP, Dagle GE, Milliman EM (1981) Chronic inhalation exposure of hamsters to nickel-enriched fly ash. Environ Res 26: 195-216

lesterol clefts occur only at very high exposure levels (Fig.229 and 230). Occasionally, alveoli in these hamsters develop vesicular emphysema.

Ultrastructure

The unique feature of asbestosis is the appearance of ferruginous bodies. Formation of these bodies has been previously described in hamsters by Suzuki and Churg (1969). Phagocytosis of small fragments of asbestos occurs primarily in alveolar macrophages. This is followed by the appearance of hemosiderin in the cytoplasm, then by intracellular transport of iron micelles from hemosiderin granules into the phagosomes with the asbestos fibers. Progressive concentration of the iron micelles occurs in the vicinity of the fibers. The central fiber, with its coating of hemosiderin, and the investing membranes of the phagosome are regarded as essential elements of the ferruginous body (Fig. 213).


The presence of ferruginous bodies is characteristic of asbestosis in hamsters and serves to distinguish it from inflammatory lesions due to other causes.

Biologic Features

Natural History. Hamsters exposed to asbestos dust (-25 ~g/I) 7 h/day, 5 days/week for 11 months experienced respiratory distress which

184 G.E.Dagle and A.P. Wehner

Fig. 226. Peribronchial distribution of macrophages and interstitial reaction after inhalation of asbestos. Hand E, x 80

forced discontinuation of the exposures (Wehner et al. 1975, 1978). Body weight and survival time were significantly decreased at these high dosage levels.

Pathogenesis. Phagocytosis of inhaled asbestos fibers by alveolar macrophages is the initial event. This is followed by an influx of additional mature macrophages into alveoli, from which they are usually cleared by the mucociliary escalator. Those macro phages that have phagocytosed longer fibers tend to remain in the alveoli longer. Longer fibers are associated with more fibrosis. The macrophages have a significant role in fibrogenesis, perhaps through the regurgitation oflysosomal enzymes during phagocytosis, but the pathogenesis is unclear (Parkes 1982).

Etiology. Several forms of asbestos, chrysotile, crocidolite, amosite, and anthophylite are all capable of inducing asbestosis. Chrysotile was used to produce the lesions in hamsters described in this report.

Fig. 227. Asbestos body in alveolar macrophage. Hand E, x 512

Frequency. Alveolar macrophages and ferruginous bodies are present in lungs of animals exposed to asbestos. Asbestosis develops in all animals exposed to asbestos.


Asbestosis in humans is characterized as bilateral, diffuse, interstitial pulmonary fibrosis and is found in persons occupationally exposed, thus affecting miners, insulators, and shipyard workers. The disease in man results in more severe, diffuse pulmonary fibrosis than is observed experimentally in hamsters. Other disorders considered to be related to asbestos exposure in humans, but not documented in hamsters, include hyaline plaques of parietal pleura, carcinoma of the lung, and skin corns (Parkes 1982). Asbestos-induced mesotheliomas of pleura and peritoneum in hamsters are discussed on page 131. The Stanton hypothesis relates longer fiber length to increased carcinogenicity of asbestos (Harington 1981).

Fig.228. Diffuse granulomatous reaction with numerous asbestos bodies, after exposure to high levels of inhaled asbestos. Hand E, x 320

Asbestos inhalation in rats produces an initial increase in the number of alveolar macrophages, followed by diffuse interstitial pulmonary fibrosis with prominent hyperplasia of alveolar epithelial cells, several kinds of peripheral lung tumors (primarily adenomas, adenocarcinomas, and squamous cell carcinomas), and mesotheliomas (Wagner et al. 1974). Noteworthy differences in experimental lesions in the rat, compared to the hamster, are the absence of ferruginous bodies and the presence of malignant pulmonary tumors.

Asbestosis, Hamster 185

Fig.229 (Above). Proliferation of bronchiolar epithelium associated with granulomatous reaction after exposure to high levels of inhaled asbestos. Hand E, x 320

Fig.230 (Below). Cholesterol clefts associated with granulomatous reaction following high levels of inhaled asbestos. Hand E, x 125

186 Alexander Kast

Fig.231. Asbestos body after inhalation of an asbestos-cement mixture. TEM, bar = l!lm

References

Harington JS (1981) Fiber carcinogenesis: epidemiologic observations and the Stanton hypothesis. JNCI 67: 977-989

Parkes WR (1982) Occupational lung disorders, 2nd edn. Butterworth,London

Suzuki Y, Churg J (1969) Structure and development of the asbestos body. Am J Pathol55: 79-107

Pulmonary Hair Embolism, Rat

Alexander Kast

Synonyms. Hair fragment emboli in the pulmonary vascular system; pulmonary embolism caused by hairs; microembolic pulmonary lesions; skin emboli in lungs.

Wagner JC, Berry G, Skidmore JW, Timbrell V (1974) The effects of the inhalation of asbestos in rats. Br J Cancer 29:252-269

Wehner AP, Busch RH, Olson RJ, Craig DK (1975) Chronic inhalation of asbestos and cigarette smoke by hamsters. Environ Res 10: 368-383

Wehner AP, Dagle GE, Cannon WC, Buschbom RL (1978) Asbestos cement dust inhalation by hamsters. Environ Res 17: 367-389

Gross Appearance

Pulmonary hair emboli are not visible grossly. Yamamoto et al. (1982), using a stereomicroscope, have seen the foreign bodies from the cut surface of rat lungs.


In histologic sections of lungs, foreign body granulomas are seen in medium-sized pulmonary arteries, interalveolar capillaries, and alveoli. In the center of these lesions, fragments of skin, hairs, and broken-off shafts of hair follicles, cut transversely, obliquely, or longitudinally, may be found. Identification of the foreign bodies is facilitated by the use of azan-Mallory stain and periodic ac-

Fig.232 (Above). Transverse section of an embolic piece of hair embedded in thrombotic material in a pulmonary artery of a male rat killed after 4 weeks of daily injections into caudal veins. Hand E, x 200 (reduced by 20%)

Pulmonary Hair Embolism, Rat 187

id-Schiff reagent (Yamamoto et al. 1982). Under polarized light this material has the same birefringent character as epidermal keratin (Innes et al. 1958; Tekeli 1974). The hair shafts have bright yellow dichroism in polarized light and with azan they appear as a red and blue double-ring structure (Schneider and Pappritz 1976). Since the structure of the hair is often unmistakably visible in hematoxylin-and-eosin-stained sections, no special methods are necessary to identify these structures (Figs. 232-234).

Fig.233 (Below). Pigmented hair segment with distinctly visible medulla cells in a thrombotic pulmonary artery of a female rabbit given 28 intravenous injections into ear veins. Hand E, x 200 (reduced by 20%)

188 Alexander Kast

From our experiences, giant cell formation is just beginning on the 3rd day after injection. At this time, the embolic hair particles are surrounded by multinucleated giant cells, fibrin, and leukocytes. At the end of subacute toxicological experiments (usually 28 days) in which material is injected intravenously, most granulomas are found in alveoli or alveolar capillaries. Less commonly, the hair particles are lodged in parietal thrombi in pulmonary vessels, where they cause partial or complete obturation of the lumen. Endovascular and perivascular inflammatory cell infiltration of vessels containing thrombi has been observed (Tekeli 1974; Yamamoto et al. 1982). Long-standing intravascular obturations become rechanneled in varying degrees in most cases. The granulation tissue often extends into the arterial wall, leading to its destruction and consequent periarterial granuloma (Schneider and Pappritz 1976). It is also striking that hair particles are often found embedded in the wall or in alveoli near to pulmonary arteries (Tsunenari and Kast 1977). This phenomenon has been explained by Johnston et al. (1981) on the basis of the ability of the macrophages to carry the foreign body through the vessel wall to reach the alveolar spaces. Infrequently, some arteries become surrounded by an abscess (Yamamoto et al. 1982). Although the injected parts of the epidermis are naturally populated with bacteria, the thromboemboli very seldom result in bacterial infection. In only six

caudal thrombi or pulmonary emboli found among 1422 rats studied (Table 19) was purulent inflammation present in adjacent tissue. No pulmonary infarction has been observed. Only one exception is present in the literature: areas of hemorrhagic infarction were described in a rabbit experimentally injected with a suspension of hair (Innes et al. 1958).


Problems in differential diagnosis may arise if no hair particles are detected within a granuloma or if the granulomatous processes are extensive. In such cases drug-specific effects may be simulated. Parasitic nodules are to be differentiated in dogs (Schneider and Pappritz 1976). The virogenic giant cell pneumonia, so-called Hecht's pneumonia (Sedlmeier and Schiefer 1971; Giese 1974), may be considered. Nests of foam cells in alveoli of rat lungs are common but seem to be involved in lipid metabolism (Flodh et al. 1974) (see page 169). In none of these lesions are the granulomas found in blood vessels. Inhaled foreign bodies, such as particles of hairs or plants, may lead to similar pulmonary granulomas, including giant cell formation (Fig. 235). For example, Sahu et al. (1975) have reported giant cells in lungs of mice 30 days after the inhalation of asbestos.

Table 19. Lesions in tail veins and lungs in 1422 Sprague-Dawley rats following daily intravenous injections for 28 days (Kast and Tsunenari 1983)

Organ Finding Males Females

Mean±SDa Total (%)b Mean±SCa Total (%)b

Tail veins Total 7.19±2.69 374 (53.2) 7.79±2.48 405 (56.3) Bleeding 2.12±1.91 110 (15.6) 1.81 ±1.58 94 (13.1) Periphlebitis 2.56±1.88 133 (18.9) 4.27±2.13 222 (30.9) Phlebitis 1.58±1.47 82 (11.7) 2.60±1.91 135 (18.8) Swelling of intima 1.38± 1.29 72 (10.2) 1.27 ± 1.09 66 (9.2) Hair in vessel wall 2.10±1.26 109 (15.5) 2.12±1.40 110 (15.3) Thrombophlebitis 3.13±2.32 163 (23.2) 3.69 ± 2.22 192 (26.7)

With hair particle 1.37 ± 1.34 71 (10.1) 1.40±1.30 73 (10.2)

Lungs Total 3.75±2.46 195 (27.7) 3.58±2.49 186 (25.9) Thromboarteritis 0.56±0.87 29 (4.1) 0.42±0.61 22 (3.1)

With hair particle 0.42 ± 0.70 22 (3.1) 0.27 ± 0.49 14 (1.9) With skin particle 0.08 ± 0.27 4 (0.6) 0.06 ± 0.24 3 (0.4)

Giant cell granulomac 3.35 ± 2.35 174 (24.8) 3.29 ± 2.43 171 (23.8) With hair particle 1.96±1.80 102 (14.5) 1.96±2.10 102 (14.2) With skin particle 0.12±0.38 6 (0.9) 0.06 ± 0.24 3 (0.4)

a Mean number of lesions in each group of 15 rats, excluding animals which did not receive all 28 injections b Total number of males = 703, of females = 719 C In alveolar capillaries or alveoli


Fig.234. Pulmonary artery of a beagle with embolic hair trapped in a thrombus. Note the concentric layers of the hair. Azan, x 200 (reduced by 15%). Schneider and Pappritz 1976)

Fig.235. Granuloma with foreign body giant cells phagocytosing inhaled particles of plants in pulmonary alveolus of a 2-year-old female rat. Hand E, x 100 (reduced by 15%)

Many reports have accumulated in the literature about pulmonary embolism and foreign body granulomatosis in man and experimental animals caused by the intravenous injection of particles such as cotton wool fibers, microcrystalline cellu-

lose, cornstarch, talc, rubber, metal or glass, and other materials (Simpson 1922; Easton 1952; Garvan and Gunner 1964; Zientara and Moore 1970; Bowden 1971; Johnston and Waisman 1971; Johnston et al. 1981; Tomashefski et al. 1981).

190 Alexander Kast

Fig.236. Sections of hair embedded in parietal thrombus in the caudal vein of a male rat. Hand E, x 200 (reduced by 15%)

Biologic Features

Pathogenesis. Intravenous injection into caudal veins of rats and mice, veins of ear of rabbits, and into veins of bigger mammals, such as dog and man, is often followed by embolism of skin particles and hair fragments. These may become part of a thrombus at the site of injection or be caught by the pulmonary filter, phagocytosed, and removed by foreign body giant cells within a few weeks. Eventually, the integrity of the affected pulmonary arteries and alveoli is restored. The entire process belongs to the so-called "pathology of therapy." In one study, we examined histologic sections of lung and caudal vein from each of 1422 rats that had received 28 daily intravenous injections of various test substances. Lesions in caudal veins were found at the site of injection in more than 50% of the rats (Table 19). These lesions included bleeding into the surrounding tissue, periphlebitis and phlebitis, cushion-like swellings of the intima, and thrombosis. The frequency of hair-containing thrombi does not significantly differ between rats injected intravenously with various test substances at different dosages and their saline-treated controls. Therefore, controls and treated animals are shown together in Table 19.

The current view is that particles of hair, hair follicles, or other bits of skin are punched out by the injecting needle and tranferred through the needle into the wall or lumen of the caudal veins. At this site they may become embedded in thrombotic material (Fig.236). If not caught in a thrombus in a vein near the site of injection, hair and skin particles are floated to the lungs, where they are stopped in the pulmonary filter. Innes et al. (1958), who were first to describe this condition, also found a fragment of hair attached to the wall of the right ventricle in one mouse, and in the right atrium of another one. According to Purkiss (1975), the pulmonary capillaries have a diameter of 7-12 !-Lm, and therefore particles greater than 12!-Lm in diameter (such as cellulose fibers with average diameter of 20.5 !-Lm) would be trapped in the vascular bed of the lung. However, due to the existence of arteriovenous shunts in the lungs, not all of them are so entrapped. Such shunts should enable the emboli to escape into veins and then into the systemic circulation. In our trials (Tsunenari and Kast 1977), only intravenously injected nylon 12 globules less than 10!-Lm in diameter have floated with the blood through the lungs into spleen, liver, and kidneys. In an experimental study of these lesions, we prepared a suspension in physiological saline solu-

Fig. 237 (Above). Hair particles in pulmonary alveolus of a male rat 3 days after the intravenous injection of a suspension of hair (for methods see Fig.239). Hand E, x 400 (reduced by 20%)

tion containing about 3000 hair particles per milliliter. The hairs were taken from the backs of rats and cut into about 10-j..Lm lengths using a cryostat (Tsunenari and Kast 1977). We injected into caudal veins of each of 50 male Sprague-Dawley rats a single dose of 0.25 ml of this rat's hair suspension for each 100 g body weight (low dose). A second group of 50 males were each given 0.75 mll 100 g body weight (high dose). Ten rats of each group were killed on days 3, 7, 14, 21, and 28 after treatment and examined histologically.


Fig. 238 (Below). Giant cells phagocytosing hair particles in an alveolus 7 days after the intravenous injection. Hand E, x 400

The development during the first 3 days of pulmonary granulomas that result from hair emboli after intravenous injection has not been described. Therefore, a quotation from Johnston et al. (1981) in studies of pulmonary embolism in the rat produced by cotton fibers is of interest: "within an hour the fibres become covered by platelets and plasma proteins and neutrophil polymorphs are attracted around them. By 2 hours macrophages congregate around the emboli and, with accumulation of epitheloid cells, granulomas are

192 Alexander Kast

20 a

18

16

14

12

10

8

6

4

2

o 3 days 28

Fig. 239. Average numbers of hair-phagocytosing giant cells in lungs of rats 3, 7, 14, 21, and 28 days after a single intravenous injection (0.75 ml/l00 g body weight) of a suspension containing about 3000 hair particles per milliliter cut at about lO!lm length. Right lung: a, apical lobe; b, cardiac lobe; c, diaphragmatic lobe; and d, azygos lobe; L, left lung. (Tsunenari and Kast 1977)

formed. By 16 hours these have increased in size with the appearance of foreign body giant cells, and reach their maximum size in 3 days." In our experiments, the frequency of embolic hair fragments in lungs was dose-dependent, and most of the fragments were observed in the groups killed 3 days after the injection. The fragments at this time were located in arteries or alveolar capillaries with little adjacent leukocytic reaction, including eosinophils, but in several cases the hair fragments were already surrounded by multinucleated giant cells (Fig. 237). Giant cells sometimes appeared to originate from the endothelium of the involved capillaries. After 7 days, phagocytosis of the bits of hair is complete (Fig. 238). During the following weeks the granulomas decline in size, the hair particles and giant cells disappear gradually, and finally, after 28 days, the lungs are essentially free of lesions (Fig.239). No hair particles were found in other organs. This confirms the statement of TekeIi (1974), who searched for but did not find keratin, fragments of hair, or hair follicles in the vessels of other organs.

Frequency. In our study of 1422 rats that had been injected intravenously with various materials in toxicology tests (Table 19), lesions in the lungs were observed in 381 of the rats (27%). These lesions consisted of giant cell granulomas with phagocytosed hair in alveoli, and hair-containing thrombi in pulmonary blood vessels (Fig. 232). No differences were attributable to the sex of the ani-

mals. The frequency is surprisingly high if one considers that only one cross section of about 3 11m thickness from one lobe of the lungs was studied in each animal. Schneider and Pappritz (1976) have pointed out that this frequency in such a small random sample of lung indicates the large number of hair particles that must have been injected in the course of the experiments. Innes et al. (1958) found 22 pulmonary granulomas (28%) (six containing foreign bodies) in 80 mice dosed intravenously at different periods from 5 through 20 days. Yamamoto et al. (1982) treated their 165 rats intravenously for 6 months and observed lesions in 28%, including such histopathologic changes as granuloma (23%), thrombus (18%), giant cells (10%), and arterial abscess (4%). Tekeli (1974) has seen pulmonary embolism after 7-30 days of treatment in 26 of his 120 rats (22% ). No difference has been demonstrated in the frequency of pulmonary embolism between carriertreated controls and test animals given various doses of injectable solutions (Innes et al. 1958; Liehn and Dahme 1975; Yamamoto et al. 1982; Kast and Tsunenari 1983). The more often the animals have been injected, the more numerous are the lesions (Innes et al. 1958); however, reparability of the lesions has to be taken into consideration.

Reparability. In our experiments with the injection of hair suspensions (Tsunenari and Kast 1977), phagocytosis has been observed rapidly progressing to nearly complete removal of the embolic hair particles within 4 weeks (Fig. 239). However, in our subacute toxicity studies, phagocytosis and removal of hairs inoculated through a needle into caudal veins was not complete after 6 weeks of recovery. The number of hair particles remaining in the lungs was significant (Table 20, Fig. 240). This suggests differences in the condition of immunity in individual rats. However, the effects are similar when different suspensions are used, one containing the rat's own hair and another made up of foreign hairs (Kast and Tsunenari 1983). Possibly, the hair of the back of the rat, used in our experiments, is more easily phagocytosed than the caudal (tail) bristles with their thicker cortex. According to Liehn and Dahme (1975), pulmonary hair emboli in their studies disappear within 8-10 weeks after the final intravenous injection. Contrary to the views of Hottendorf and Hirth (1974) and Schneider and Pappritz (1976), who believe that the risk is minor if the hairs at the in-


Fig. 240. Embolic hair fragment distinctly affected by phagocytosis but remaining in lung 6 weeks after the last of 28 daily intravenous injections, male rat. Hand E, x 200 (reduced by 15%)

Table 20. Lesions in tail veins and lungs of90 Sprague-Dawley rats injected intravenously daily for 28 days and allowed to recover for 6 weeks (Kast and Tsunenari 1983)

Organ Finding

Tail veins Total Bleeding Periphlebitis Phlebitis Swelling of intima Hair in vessel wall Thrombophlebitis

With hair Lungs Total

Thromboarteritis With hair particle With skin particle

Giant cell granulomac

With hair particle With skin particle

Males

Mean±SDa

3.60±1.52 o 0.40 ± 0.55 1.00± 1.22 0.40±0.55 1.40±1.34 1.80± 1.79 0.60±0.55 0.80±0.84

} None

0.80±0.84 0.40±0.55 None

Total (%)b

18 (38.3) o (0) 2 (4.3) 5 (10.6) 2 (4.3) 7 (14.9) 9 (19.1) 3 (6.4) 4 (8.5)

4 (8.5) 2 (4.3)

Females

Mean±SDa

3.20±1.92 0 0 0.80±1.30 0.40±0.55 1.40±1.14 1.60± 1.52 0.80±1.10 0.20 ± 0.45

0.20±0.45 0.20 ± 0.45

Total (%)b

16 (37.2) o (0) o (0) 4 (9.3) 2 (4.7) 7 (16.3) 8 (18.6) 4 (9.3) 1 (2.3)

1 (2.3) 1 (2.3)

a Mean number oflesions in each group often rats excluding animals which did not receive all 28 injections b Total number of males =47, of females =43 C In alveolar capillaries or alveoli

jection site are carefully clipped and the injection is carried out with care, we agree with other authors that no matter how carefully the intravenous injection is done, pulmonary pathological changes are inevitable results of intravenous injection!


In man, three single observations of skin embolism have been published by Hirst and Toyama, (1968), Andrew (1976), and Gilbert and Borchard (1980). The skin particles in these cases were be-

194 Alexander Kast

lieved to have been punched out by puncture of the cephalic vein or heart and carried by the blood to the lungs. A similar but very rare finding is known in human pathology: after amniotic fluid embolism, small lanugo hairs of the human fetus are occasionally found in capillaries of maternal lungs (Scotti 1966; Adamsons et al. 1971; Morgan 1979). According to Schneider and Pappritz (1976), the dermal fibrosis at the point of repeated injections in beagle dogs (cephalic vein) forces many hair roots into deeper skin layers, making them more vulnerable for an excision by the needle and transfer into the puncture channel or the vein's wall. These authors identified hair particles within the arteries in ten of their 30 dogs (33%) treated over 4 weeks. In the studies of Hottendorf and Hirth (1974), eight of 67 beagles (12%) had granulomas in small pulmonary arteries. Among 64 Himalayan rabbits treated intravenously over 4 weeks, Kast and Tsunenari (1983) discovered five giant cell granulomas, two arterial thrombi (Fig. 233), and one fat cell embolus of the lungs (13%).

References

Adamsons K, Mueller-Heubach E, Myers RE (1971) The innocuousness of amniotic fluid infusion in the pregnant rhesus monkey. Am J Obstet Gynecol 109: 977-984

Andrew IH (1976) Pulmonary skin embolism: a case report. Pathology 8: 185-187

Bowden DH (1971) The alveolar macrophage. Curr Top Pathol55: 1-36

Easton TW (1952) The role of macrophage movements in the transport and elimination of intravenous thorium dioxide in mice. Am 1 Anat 90: 1-33

F10dh H, Magnusson G, Magnusson 0 (1974) Pulmonary foam cells in rats of different age. Z Versuchstierkd 16: 299-312

Garvan 1M, Gunner BW (1964) The harmful effects of particles in intravenous fluids. Med J Aust 51: 1-6

Giese W (1974) Akute interstitielle Pneumonien. In: Doerr W (ed) Organpathologie, vol 1. Thieme, Stuttgart, chap 3

Gilbert P, Borchard F (1980) Hautembolie der Lunge. Pathologe 1: 161-163

HirstAE, ToyamaM (1968) Skin embolism to the lung. lAMA 205: 54

HottendorfGH, Hirth RS (1974) Lesions of spontaneous subclinical disease in beagle dogs. Vet Pathol 11: 240-258

Innes lRM, Donati El, Yevich PP (1958) Pulmonary lesions in mice due to fragments of hair, epidermis and extraneous matter accidentally injected in toxicity experiments. Am 1 Pathol34: 161-167

10hnston WH, Waismanl (1971) Pulmonary com starch granulomas in a drug user. Arch Pathol92: 196-202

10hnston B, Smith P, Heath D (1981) Experimental cottonfibre pulmonary embolism in the rat. Thorax 36: 910-916

Kast A, Tsunenari Y (1983) Hair embolism in lungs of rat and rabbit caused by intravenous injection. Lab Anim 17: 203-207

Liehn HD, Dahme E (1975) Mikroembolische Lungenveranderungen bei Versuchstieren nach intravenoser Applikation. Med Int Congr. Proceedings of the European Society of Toxicology, Series no 345: 319-323

Morgan M (1979) Amniotic fluid embolism. Anaesthesia 34:20-32

Purkiss R (1975) Effects and distribution of intravenously administered cellulose particles in mice. 1 Pharm Pharmacol 27: 290-292

Sahu AP, Dogra RK, Shanker R, Zaidi S (1975) Fibrogenic response in murine lungs to asbestos. Exp Pathol 11: 21-24

Schneider P, Pappritz G (1976) Hairs causing pulmonary emboli. A rare complication in long-term intravenous studies in dogs. Vet Pathol 13: 394-400

Scotti TM (1966) Disturbance of body water and electrolytes, and of circulation of blood. In: Anderson W (ed) Pathology (Asian edition). Mosby, St. Louis, chap 4

Sedlmeier H, Schiefer B (1971) Entziindungen der Lunge. In: Dobberstein 1, Pallaske G, Stuenzi H (eds) Handbuch der speziellen pathologischen Anatomie der Haustiere, vol 7. Parey, Berlin

Simpson M (1922) The experimental production of macrophages in the circulating blood. 1 Med Res 43: 77 -144

Tekeli S (1974) Occurrence of hair-fragment emboli in the pulmonary vascular system of rats. Vet Pathol 11: 482-485

Tomashefski JF Jr, Hirsch CS, lolly PN (1981) Microcrystalline cellulose pulmonary embolism and granulomatosis. Arch Pathol Lab Med 105: 89-93

Tsunenari Y, Kast A (1977) Hair embolism in lungs of rat after intravenous injection into caudal veins. Arzneim Forsch 27: 1979-1982

Yamamoto H, Imai S, Okuyama T, Tsubura Y (1982) Pulmonary lesions in rats caused by intravenous injection. Acta Pathol Ipn 32: 741-747

Zientara M, Moore S (1970) Fatal talc embolism in a drug addict. Hum Pathol1: 324-327

LESIONS DUE TO INFECTION

Sendai Virus Infection, Lung, Mouse and Rat

David G. Brownstein

Synonyms. Hemagglutinating virus infection of Japan (HVJ); Japanese hemagglutinating virus infection (JHV); hemagglutinating virus infection of the mouse (HVM); parainfluenza virus infection.

Gross Appearance

Mice. Asymptomatic or mildly affected mice usually have no gross pulmonary changes other than increased lung weights, although a few mildly affected mice have white lines along the course of bronchi, white foci in lymph nodes, and dual lobes of consolidation. Average increases of 50% have been reported by the 5th day and 100% by the 14th day after experimental infection of Swiss mice (Robinson et al. 1968). Overtly ill and genetically susceptible mice have more dramatic increases in lung weights (200%-300%) (Parker et al. 1978). In these cases, one or more lung lobes are plum-colored or contain sharply demarcated plum-colored foci which exude frothy sanguinous fluid when cut. If these mice survive into the 3rd week, consolidated foci are gray. Extrapulmonary changes reflect local and systemic activation of the immune system and stress. Regionallymph nodes enlarge during the 2nd week, a change most obvious in the cervical lymph nodes. These nodes may triple or quadruple in weight. Splenic enlargement (20%-50% in weight) also occurs during the 2nd week. Thymic involution is a stress-related change, the degree being roughly porportional to the degree of lung parenchymal injury.

Rats. Asymptomatic infections are the rule and follow a pattern similar to that described for asymptomatic or mildly affected mice.


Mice. Histopathologic changes in experimental and natural disease can be divided into: acute phase, characterized by degeneration and necrosis of target epithelium and exudative inflammation; reparative phase, characterized by regeneration of damaged target cells and interstitial inflammation; and resolution phase, characterized by a rapid subsidence of inflammation and repair. During the acute phase, which lasts from 8 to 12 days, there is a descending infection of conducting airway epithelium. This frequently extends to proximal type II and, to a lesser degree, type I alveolar epithelium (Brownstein et al. 1981; Parker and Richter 1982; Richter 1970, 1973; Zurcher et al. 1977). The result is an acute endobronchitis-bronchiolitis and bronchogenic alveolitis. The earliest airway changes are segmental. Infected bronchiolar epithelial cells are hypertrophied with eosinophilic foamy, granular, or homogeneous cytoplasm. The nuclei of infected cells are poorly polarized and may be enlarged and vesicular. Inflammatory infiltrates appear shortly after these initial cytological changes (Fig. 241). The lamina propria and adventitia are expanded by edema, dilated lymphatics, and cellular infiltrates. Initially, these infiltrates are a mixture of polymorphonuclear leukocytes, reactive lymphoid cells with moderate amounts of homogeneous amphophilic or eosinophilic cytoplasm, and large round or fusiform lymphoreticular cells. The relative number of each type depends on the age and genetic composition of the host and the dose and route (intranasal versus aerosol) of viral exposure. In genetically susceptible or immature mice, neutrophils often predominate as they do after exposure to large doses of virus. Peribronchiolar pulmonary arterioles and venules have increased intimal cellularity. The endothelium is hypertrophied and elevated by asymmetric accumulations of leukocytes in the


Fig. 241 (Above). Sendai virus infection, acute phase. Bronchiolar epithelium is hypertrophied with some loss of nuclear polarity. The adventitia is edematous and infiltrated with lymphoid cells. Hand E, x 354 (reduced by 15%)

subendothelial layer of the intima. There is striking leukocyte pavementing. The adventitia of these vessels is also hypercellular due to leukocytic infiltrates similar to those infiltrating airways. The adventitia is also expanded by edema. Cytopathic changes in bronchiolar epithelium are afforded some specificity if syncytia, cytoplasmic

Fig.242 (Below). Sendai virus infection, acute phase. Bronchiole with multiple epithelial syncytia. Hand E, x 791 (reduced by 15%)

inclusions, or intranuclear inclusions are present. The lesions may depend, in part, on virus and mouse strain. These changes are usually not seen or are equivocal, since infected cells tend to slough prior to their development. Syncytia usually appear as central clusters of condensed or pyknotic epithe-

Fig. 243 (Above). Sendai virus infection, acute phase. Alveoli radiating from an infected terminal bronchiole contain a mixed inflammatory exudate. Alveolar septa are edematous and infiltrated with mononuclear cells. Hand E, x 345 (reduced by 15%)

lial nuclei surrounded by foamy eosinophilic cytoplasm that crowds adjacent cells (Fig. 242). Cytoplasmic inclusions represent aggregates of redundant nucleocapsids within the cytoplasmic matrix (see Ultrastructure). They are most obvious as homogeneous spherical or irregular eo-

Sendai Virus Infection, Lung, Mouse and Rat 197

Fig.244 (Below). Sendai virus infection, reparative phase. Bronchiole with disorganized regenerating epithelium. Nuclei are pleomorphic and vesicular with prominent nucleoli. Hand E, x 245 (reduced by 15%)

sinophilic cytoplasmic bodies surrounded by a narrow halo. This halo is a fixation shrinkage artifact. Intranuclear inclusions are rare in Sendai virus infections but intranuclear viral particles have been described in DBAI2 mice (Richter 1970) and intranuclear inclusions have been

198 David G.Brownstein

reported in athymic nude mice (Ward et al. 1976). Near the end ofthe acute phase, infected epithelium has sloughed in sheets or as individual cells which lie within the lumen along with an inflammatory exudate. Because of the segmental nature of the infection, some airways may be devoid of lining cells while others are lined by intact, hypertrophied epithelium. Airways with desquamating epithelium frequently have focally dense lymphoid aggregates within the lamina propria, which cause overlying epithelium to bulge into the lumen. The alveolar component of the acute phase is characterized by accumulations of polymorphonuclear leukocytes, macrophages, lymphoid cells, and desquamated pneumocytes within alveolar spaces. Alveolar septa are thickened by edema, congested capillaries, and hypertrophied alveolar comer cells. These changes are multifocal, being oriented around infected terminal bronchioles. In severe cases foci of parenchymal inflammation may become confluent, resulting in lobar pneumonia. Alveolar spaces may fill with fibrin or extravasated blood. Foci of emphysema may occur where septal integrity has been destroyed. Near the end of the acute phase fibrin deposits have condensed along denuded alveolar surfaces or as discrete deposits in alveolar spaces. Increased cellularity of thickened alveolar septa is due to accumulating mononuclear cells (Fig. 243). Inflamed alveoli may be partially atelectatic. The reparative phase is heralded by the appearance of regenerating epithelium. This may be seen as early as the 3rd day, but does not predominate over degenerative airway changes until days 8-12. Initially, partially denuded airways contain continuous or interrupted patches of low cuboidal, polygonal, or squamous cells with basophilic cytoplasm and large pleomorphic vesicular nuclei which are poorly polarized and contain prominent nucleoli (Fig.244). Mitotic figures are common in these cells. Once denuded airways are reepithelialized, lining cells become columnar and rapidly assume a normal mucociliary appearance. Sessile or pedunculated airway excrescences are extremely common during the reparative phase. These may be composed solely of epithelium or have cores of lymphoreticular cells or fibroblasts. These are ephemeral structures and are rarely seen after the 3rd week. It is common during this phase for terminal bronchioles to be lined by nonkeratinizing stratified squamous epithelium. The metaplastic epithelial cells most closely resemble spinous cells, although intercel-

lular bridges are usually difficult to identify. The adventitia and lamina propria of segments undergoing repair are less edematous than in the acute phase, and are densely infiltrated with lymphocytes and plasma cells. The intimal cellularity of pulmonary arterioles and venules seen during the acute phase in usually absent in the reparative phase, but adventitial infiltrates of lymphocytes and plasma cells are prominent. During the reparative phase, alveolar inflammation has shifted from the airspaces to the interstitium. Two forms of alveolar repair are recognized. In the first, septa are lined by cuboidal epithelium (adenomatous hyperplasia, alveolar bronchiolization, alveolar epithelialization) (Fig. 245). These epithelial cells are initially undifferentiated, but soon differentiate into pneumocytes or ciliated, mucous or Clara cells. In the second form of repair, sheets and nests of metaplastic squamous epithelium fill alveolar spaces (Fig. 246). There is partial or total atelectasis of affected alveoli during this phase. If fibrin has been deposited it undergoes lysis or organization. Macrophages surround and infiltrate large fibrin deposits. Plump fibroblasts may also traverse these deposits. During the 3rd week collagen bundles can be identified in organizing fibrin depositis. The reparative phase is usually complete by the end of the 3rd week. The resolution phase may be as short as 1 week, so that by the end of the 4th week residual lesions are difficult to identify. There are, however, several changes which may persist for long periods. The most severe sequela, and one that may persist for the life of the animal, is organizing alveolitis with or without bronchiolitis fibrosa obliterans (Fig. 247). This change is the end result of the organization of extensive fibrin deposits in denuded alveoli and terminal airways. Sheets of haphazardly arranged collagen bundles and fibroblasts in natural infections may obliterate parenchymal architecture or may fill alveolar spaces, leaving parenchymal architecture intact. Other residual lesions include foci of emphysema containing inspissated secretions, cholesterol crystals, and macrophages; focal aggregates of foamy, alveolar macrophages; focal thickening of alveolar septa; focal adenomatous hyperplasia of alveoli; and perivascular and peribronchiolar lymphoplasmacytic infiltrates. These changes have been identified in mice up to 1 year after infection with Sendai virus (Appel et al. 1971; Parker and Richter 1982; Robinson et al. 1968). Athymic nude mice, unlike euthymic mice, develop progressive lung changes due to persistent in-

Fig.245 (Above). Sendai virus infection, reparative phase. Alveoli are lined by cuboidal epithelium and septa are infiltrated with lymphoid cells. Hand E, x 600 (reduced by 15%)

fection with Sendai virus (Iwai et al. 1979; Ward et al. 1976). Pulmonary changes are most often diffuse due to extensive parenchymal involvement. Alveolar septa are thickened by edema, neutrophils, and macrophages. Alveolar epithelialization is striking and squamous metaplasia is prominent in some cases. Bronchiolar epithelium


Fig.246 (Below). Sendai virus infection, reparative phase. Alveolar squamous metaplasia. Hand E, x 600 (reduced by 15%)

is hyperplastic with numerous mitotic figures. Airways may be filled with neutrophils. Lymphoid infiltrates are usually sparse.

Rats. Information on the microscopic appearance of Sendai-virus-induced lung lesions in rats is limited. Lesions in a colony of mixed rat strains that


Fig. 247. Sendai virus infection, resolution phase. Organizing alveolitis. Hand E, x 244 (reduced by 15%)

develop antibodies to Sendai virus have been reported but virus isolation was not attempted (Burek et al. 1977). These rats had mild to severe peribronchial and perivascular cuffing by lymphocytes and plasma cells, and interstitial pneumonia. Bronchiolar epithelium was hyperplastic and focally ulcerated. Unfortunately, the rats also developed antibodies to pneumonia virus of mice. In the author's experience this virus is capable of producing inflammatory lung disease in the rat. Necrotizing bronchitis has been reported in germfree rats inoculated intranasally with Sendai virus (Jacoby et al. 1979).

Ultrastructure

Limited ultrastructural pathology has been reported of experimental and spontaneous Sendai virus infections in euthymic mice (Brownstein et al. 1981; Parker and Richter 1982; Richter 1970, 1973; Zurcher et al. 1977). The ultrastructure of naturally infected athymic nude mice has also been reported (Ward et al. 1976). The primary site of replication is the epithelium of bronchi and bronchioles and it is within ciliated and Clara cells that ultrastructural evidence of replication is most easily seen. Nucleocapsid assembly occurs in the cytosol at a rate far in excess of the rate of virus assembly. The result is massive accumula-

tions of nucleocapsids, which appear as poorly delineated aggregates or crystalline arrays of rigid hollow fibrils 16-18 nm in diameter. Intranuclear aggregates of similar-appearing fibrils have been reported and these apparently correspond to the intranuclear inclusions seen by light microscopy. The significance of these aggregates is not known; they are rare and appear late in infection, suggesting that they may be transported from sites of synthesis in the cytosol (Choppin and Compans 1975). Nucleocapsids align beneath modified segments of plasma membrane to initiate exotrophy. These modified segments are thickened by glycoprotein surface spikes and an electron-dense layer on the inside of the plasma membrane. Budding forms may be spherical, filamentous, or pleomorphic (Darlington et al. 1970). This appearance of productively infected cells is also seen in infected type II alveolar epithelium (Brownstein et al. 1981). An additional cytoplasmic inclusion consisting of membrane-bound, randomly arranged hollow fibrils, 15-20 nm in diameter, surrounded by 40-nm electron-dense cuffs has been reported in these cells (Zurcher et al. 1977). Incomplete reports indicate viral components in type I alveolar epithelium and septal capillary endothelium (Parker and Richter 1982; Ward et al. 1976). Only intranuclear nucleocapsids have been described in the former (nude mice). Viral assembly has not been observed in ei-

ther cell type. Both of these may be examples of abortive infections. Alveolar macrophages are susceptible to abortive infections in vitro (Eustatia et al. 1972; Mims and Murphy 1973), but ultrastructural evidence of viral replication is lacking. The author has seen numerous virions and nucleocapsids within heterophagosomes of alveolar macrophages in experimentally infected mice. This perhaps represents phagocytosis of debris containing viral particles.


Mice. Sendai virus pneumonia must be distinguished from pneumonias caused by mouse coronaviruses, K virus, pneumonia virus, Mycoplasma pulmonis, and Corynebacterium kutscheri. The chronic wasting disease produced in athymic nude mice must be distinguished from similar syndromes caused by mouse coronarviruses, mouse adenovirus, Pneumocystis, Giardia, Spironucleus, and Toxoplasma.

Rats. Sendai virus pneumonia must be distinguished from pneumonias caused by rat coronavirus, pneumonia virus, Mycoplasma pulmonis, Streptococcus pneumoniae, and Corynebacterium kutscheri. Some pulmonary changes caused by Sendai virus infection mimic those changes caused by exposure of rodents to halogenated aromatic hydrocarbons (Reid et al. 1973), oxidant gases (Stephens et al. 1974), and other toxicants which have the terminal conducting airways as target structures.

Biologic Features

Natural History. Sendai virus causes acute limited infections in immunocompetent rodents. There is no evidence for latency or chronic infections (Fujiwara et al. 1976; van der Veen et al. 1974). Enzootic and epizootic forms exist. Enzootic infections occur in partially immune rodent colonies where susceptible individuals are regularly introduced to perpetuate the infection. This can occur in breeding or open colonies. In breeding colonies, the susceptible population is the weaning age animals (3-6 weeks), due to their declining passive immunity (Parker and Reynolds 1968). Enzootic infections are usually subclinical.


Epizootic infections occur in naive rodent colonies and either die out after 2-7 months or become enzootic if the proper conditions exist (Parker and Reynolds 1968; Parker et al. 1978). Clinical signs may be associated with epizootic infections in mice but have not been reported in rats. Such factors as strain susceptibility, age, husbandry, shipping, and copathogens are important in precipitating overt disease in mice (Jakab 1974; Jakab and Dick 1973; Parker et al. 1978; Ward 1974; Zurcher et al. 1977). Breeding colonies may exhibit neonatal or weanling mortality, prolonged gestation, fetal resorption, or runting in young mice (Bhatt and Jonas 1974; Iwai et al. 1979). Adult mice may exhibit anorexia, depression, ruffled fur, hunched posture, chattering, conjunctivitis, and photophobia. Sendai virus is transmitted by aerosol and contact routes. Naso- and oropharyngeal secretions develop high infectivity titers during the 1st week. Fifty to seventy percent of mice will become infected after 24 h of contact with a transmitter mouse (van der Veen et al. 1970, 1972). Attack rates are lower for aerosol transmission unless multiple transmitters are present (van der Veen et al. 1974). Athymic nude mice infected with Sendai virus are persistently infected. Most exhibit dramatic weight loss, depression, wrinkled skin, dyspnea, and cyanosis and usually die between 2 and 10 weeks later (Iwai et al. 1979; Ward et al. 1976).

Pathogenesis. Viral replication is restricted to the respiratory tract in natural infections, although a low-level transient viremia may occur. Peak titers are reached in 3-6 days and virus is usually not recoverable after 8-12 days (Appel et al. 1971; Parker et al. 1978; Robinson et al. 1968; Sawicki 1962; Stewart and Tucker 1978; van Nunen and van der Veen 1967). Adult mice eliminate virus earlier than suckling mice and develop lower peak titers (Sawicki 1961). Outbred mice eliminate virus earlier than inbred mice (Stewart and Tucker 1978). Seroconversion (hemagglutination inhibition, complement fixation) is usually detectable on days 7-9 (Appel et al. 1971; Robinson et al. 1968; Stewart and Tucker 1978; van Nunen and van der Veen 1967). Mice are usually leukopenic by the 7th day of infection; leukocytosis follows on days 8-11 (Robinson et al. 1968). Offspring of naturally infected dams rapidly acquire neutralizing and complement-fixing antibodies of the IgG1

and IgG2 subclasses with the onset of nursing. These titers plateau on days 7 -14 and then rapidly decline (lida et al. 1973).


Etiology. Sendai virus is a parainfluenza 1 virus of the genus Paramyxovirus, family Paramyxoviridae. It is a pleomorphic, enveloped virus measuring approximately 100-300nm in length. The helical nucleocapsid contains a continuous single strand of RNA. The envelope is studded with two types of surface-projecting glycosylated polypeptides. The larger projection has hemagglutination and neuraminidase activity; the smaller projection has hemolysin and cell fusion activity. The virus rapidly inactivates at temperatures between 20° and 37°C (Chanock et al. 1963).

Frequency. Sendai virus is ubiquitous in colonies of mice and rats. A survey of various institutional and commercial colonies, published in 1978, indicated that 66% of mouse colonies and 63% of rat colonies had experienced infections (Parker et al. 1978). Attack rates usually exceed 50% (Parker et al. 1964, 1978).


Parainfluenza viruses naturally infect humans, other primates, dogs, cattle, sheep, and birds in addition to rodents. All mammalian parainfluenza viruses replicate and cause disease primarily in the respiratory tract. They have a tropism for the epithelium of the conducting airways. Besides Sendai virus, histopathology is well described for parainfluenza 3 virus infection in calves (Dawson et al. 1965; Omar et al. 1966; Tsai and Thomson 1975). These lesions are similar to those caused by Sendai virus in rodents. Intranuclear inclusions are more prevalent and intracytoplasmic inclusions are more distinct in parainfluenza 3 virus infections.

References

Appel LH, Kovatch RM, Reddecliff 1M, Gerone PI (1971) Pathogenesis of Sendai virus infection in mice. Am 1 Vet Res 32: 1835-1841

Bhatt PN, 10nas AM (1974) An epizootic of Sendai infection with mortality in a barrier-maintained mouse colony. Am 1 Epidemiol100: 222-229

Brownstein DG, Smith AL, 10hnson EA (1981) Sendai virus infection in genetically resistant and susceptible mice. Am J Patholl05: 156-163

BureklD, ZurcherC, van Nunen MCl, HollanderCF (1977) A naturally occurring epizootic caused by Sendai virus in breeding and aging rodent colonies. II. Infection in the rat. Lab Anim Sci 27: 963-971

Chanock RN, Parrott RH, Johnson KM, Kopikian AZ,

Bell lA (1963) Myxoviruses: parainfluenza. Am 1 Respir Dis 88 (Suppl): 152-166

Choppin PW, Compans RW (1975) Reproduction of paramyxoviruses. Compr Virol4: 95-178

Darlington RW, Portner A, Kingsbury DW (1970) Sendai virus replication: an ultrastructural comparison of productive and abortive infections in avian cells. 1 Gen Viro19: 169-177

Dawson PS, Darbyshire IH, Lamont PH (1965) The inoculation of calves with parainfluenza 3 virus. Res Vet Sci 6: 108-113

Eustatia 1M, Maase E, van Heiden P, van der Veen 1 (1972) Viral replication in mouse macrophages. Arch Virusforsch 39: 376-380

Fujiwara K, Takenaka S, Shumiya S (1976) Carrier state of antibody and viruses in a mouse breeding colony persistently infected with Sendai and mouse hepatitis viruses. Lab Anim Sci 26: 153-159

Iida T, Tajima M, Murata Y (1973) Transmission of maternal antibodies to Sendai virus in mice and its significance in enzootic infection. 1 Gen Viro118: 247-254

Iwai H, Goto Y, Ueda K (1979) Response of athymic nude mice to Sendai virus. Ipn 1 Exp Med 49: 123-130

lacoby RO, Bhatt PN, 10nas AM (1979) Viral disease. In: Baker HI, Lindsey lR, Weisbroth SH (eds) The laboratory rat, vol 1, Biology and diseases. Academic, New York, chap 11

lakab Gl (1974) Effect of sequential inoculations of Sendai virus and Pasteurella pneumotropica in mice. 1 Am Vet Med Assoc 164: 723-728

lakab Gl, Dick EC (1973) Synergistic effect in viral-bacterial infection: combined infection of the murine respiratory tract with Sendai virus and Pasteurella pneumotropica. Infect Immun 8: 762-768

Mims CA, Murphy FA (1973) Parainfluenza virus Sendai infection in macrophages, ependyma, choroid plexus, vascular endothelium and respiratory tract of mice. Am 1 Pathol 70: 315-328

Omar AR, lennings AR, Betts AO (1966) The experimental disease produced in calves by the J-121 strain of parainfluenza virus type 3. Res Vet Sci 7: 379-388

Parker JC, Reynolds RK (1968) Natural history of Sendai virus infection in mice. Am J Epidemiol 88: 112-125

Parker lC, RichterCB (1982) Viral diseases of the respiratory system. In: FosterHL, SmalllD, FoxlG (eds) The mouse in biomedical research, vol 2, Diseases. Academic, New York, chap 8

Parker lC, Tennant, RW, Ward TG, Rowe WP (1964) Enzootic Sendai virus infections in mouse breeder colonies within the United States. Science 146: 936-938

Parker lC, Whiteman MD, Richter CB (1978) Susceptibility of inbred and outbred mouse strains to Sendai virus and prevalence of infection in laboratory rodents. Infect Immun 19: 123-130

Reid WD, lIett KF, Glick 1M, Krishna G (1973) Metabolism and binding of aromatic hydrocarbons in the lung. Relationship to experimental bronchiolar necrosis. Am Rev Respir Dis 107: 539-551

RichterCB (1970) Application of infectious agents to the study of lung cancer: studies on the etiology and morphogenesis of metaplastic lung lesions in mice. USAEC Symposium Series 21: 365-382

Richter CB (1973) Experimental pathology of Sendai virus infection in mice. 1 Am Vet Med Assoc 163: 1204

Robinson TWE, Cureton RJR, Heath RB (1968) The pathogenesis of Sendai virus infection in the mouse lung. J Med Microbiol 1: 89-95

Sawicki L (1961) Influence of age of mice on the recovery from experimental Sendai virus infection. Nature 192: 1258-1259

Sawicki L (1962) Studies on experimental Sendai virus infection in laboratory mice. Acta Virol (Praha) 6: 347-351

Stephens RJ, Sloan MF, Evans MJ, Freeman G (1974) Early response of lung to low levels of ozone. Am J Pathol 74:31-58

Stewart RB, Tucker MJ (1978) Infection of inbred strains of mice with Sendai virus. Can J Microbiol 24: 9-13

Tsai KS, Thomson RG (1975) Bovine parainfluenza type 3 virus infection: ultrastructural aspects of viral pathogenesis in the bovine respiratory tract. Infect Immun 11: 783-803

van Nunen MCJ, van der Veen J (1967) Experimental in-

Rat Coronavirus Infection, Lung, Rat

David G. Brownstein

Synonym. Parker's rat coronavirus infection.

Gross Appearance

Naturally infected adult rats rarely have grossly observable changes. Experimentally infected 9-10 week old axenic rats develop gross lesions in the lung on postinoculation days 6 and 7, which consist of randomly dispersed red-brown to gray foci, less than 1 mm in diameter (Bhatt and Jacoby 1977). Although rat coronavirus may cause fatal pneumonia in a high percentage of newborn and day-old rats, gross pulmonary lesions have not been described (Parker et al. 1970).


Lung changes in young adult rats are mild and short-lived (Bhatt and Jacoby 1977). Bronchus-associated lymphoid tissue is hyperplastic (Fig. 248), some pulmonary veins and venules are cuffed by lymphocytes (Fig.249), and there is patchy interstitial pneumonia (Fig. 250). Septa of affected alveoli are thickened by mononuclear cells and neutrophils. Adjacent alveolar spaces contain desquamated pneumocytes, foamy macro phages, lymphocytes, and neutrophils.

Rat Coronavirus Infection, Lung, Rat 203

fection with Sendai virus in mice. Arch Virusforsch 22: 388-397

van der Veen J, Poort Y, Birchfield DJ (1970) Experimental transmission of Sendai virus infection in mice. Arch Virusforsch 31 : 237-246

van der Veen J, Poort Y, Birchfield DJ (1972) Effect of relative humidity on experimental transmission of Sendai virus in mice. Proc Soc Exp Bioi Med 140: 1437-1440

van der Veen J, Poort Y, Birchfield DJ (1974) Study of the possible persistence of Sendai virus in mice. Lab Anim Sci 24: 48-50

Ward JM (1974) Naturally occurring Sendai virus disease of mice. Lab Anim Sci 24: 938-942

WardJM, HouchensDP, CollinsMJ, YoungDM, Reagan RL (1976) Naturally-occurring Sendai virus infection of athymic nude mice. Vet Pathol13: 36-46

Zurcher C, Burek JD, van Nunen MCJ, Meihuizen SP (1977) A naturally occurring epizootic caused by Sendai virus in breeding and aging rodent colonies. I. Infection in the mouse. Lab Anim Sci 27: 955-962

Transient rhinotracheitis also occurs and may lead to segmental erosion of the respiratory epithelium covering nasal turbinates. The lamina propria is edematous and infiltrated with lymphocytes and neutrophils. Some nasal respiratory surfaces are covered with exudate consisting of mucus, neutrophils, desquamated epithelium, and detritus. Tracheal epithelium is rarely eroded but large numbers of trans epithelial neutrophils may be present. The lamina propria is mildly edematous, congested, and infiltrated with lymphocytes and neutrophils. Lesions of salivary glands are uncommon but may help distinguish rat coronavirus infections from other rat respiratory infections. Mild parotitis and submaxillary sialoadenitis have been reported and are identical to, but less severe than, those caused by sialodacryoadenitis virus (Bhatt and Jacoby 1977). There is necrosis of salivary ducts with periductular and interstitial inflammatory edema. Lesions apparently do not occur in lacrimal glands. Infected neonatal rats develop diffuse interstitial pneumonia, focal atelectasis, and compensatory emphysema (Parker et al. 1970).


Ultrastructure

Infected epithelial cells have focally dilated cisternae of endoplasmic reticulum and cytoplasmic vacuoles which contain spherical dense cores, 60-70 nm, in diameter, surrounded by an envelope 80-120 nm in diameter (Parker et al. 1970; Jonas et al. 1969). The characteristic corona, seen in negatively stained preparations, is not seen by transmission ultramicroscopy. Morphologically, rat coronavirus is indistinguishable from sialodacryoadenitis virus.


Respiratory tract lesions must be differentiated from those caused by sialodacryoadenitis virus, Sendai virus, pneumonia virus, Mycoplasma pulmonis, and pathogenic bacteria. Lesions in salivary glands, when present, are generally milder than those caused by sialodacryoadenitis virus.

Biologic Features

Natural History. Rat coronavirus causes acute limited infections of the respiratory tract. The epizootiologic characteristic of this infection has not been reported. No evidence for a carrier state has been reported and host range studies have been limited to the rat. The virus is highly infectious and is presumably transmitted by aerosol or direct contact. The infection is subclinical in rats shortly following weaning. Infected neonatal rats may die with severe respiratory distress.

PathogenesiS. Rat coronavirus is epitheliotropic and replicates at all levels of the respiratory tract during the 1st week of infection. The highest titers

<l Fig.248 (Above). Rat coronavirus infection, lung. Bronchiole with mildly hyperplastic lymphoid nodules following experimental infection. Hand E, x 59 (reduced by 15%)

Fig. 249 (Middle). Rat coronavirus infection, lung. Perivenular lymphoid cells in an experimentally infected rat. H and E, x 536 (reduced by 15%)

Fig.250 (Below). Inerstitial pneumonia following experimental infection with rat coronavirus. Alveoli contain foamy macrophages and lymphoid cells. Septa are infiltrated with mononuclear cells. Hand E, x 536 (reduced by 15%)

Rat Coronavirus Infection, Lung, Rat 205

are reached in the nasal cavity and trachea. limited replication occurs in salivary tissues. Neutralizing antibodies to rat coronavirus and sialodacryoadenitis virus are detectable on day 6 or 7. Complement-fixing antibodies appear later and cross-react with sialodacryoadenitis and mouse hepatitis viral antigens (Bhatt and Jacoby 1977; Parker et al. 1970; Jacoby et al. 1979).

Etiology. Rat coronavirus is a typical member of the Coronaviridae: a pleomorphic, enveloped RNA virus with plump, pedunculated surface projections (corona). It measures 76-98 nm in diameter in negatively stained preparations with 12-25 nm surface projections (Parker et al. 1970). Virions are formed in cytoplasmic vesicles and cisternae of endoplasmic reticulum. The virus is closely related antigenically to sialodacryoadenitis virus (Bhatt et al. 1972).

Frequency. The frequency of rat coronavirus infection within rat colonies is difficult to ascertain because of the close relationship of this virus to sialodacryoadenitis virus. Serological evidence of infection with coronaviruses is common in commercial and institutional rat colonies. In one survey of 4 germ-free, 5 specific-pathogen-free (SPF), and 11 conventional rat colonies, 3 of the SPF and 11 of the conventional colonies were infected while the germ-free colonies were not (Parker et al. 1970). In another report, all retired breeders from six vendors were positive (Jacoby et al. 1979).


Coronaviruses are ubiquitous in humans, animals, and birds (Bohl 1981). They cause enteritis in swine, cattle, dogs, mice, turkeys, and humans; encephalomyelitis in swine and mice; systemic infections in cats and mice; sialodacryoadenitis in rats; and respiratory infections in chickens, rats, and humans. Avian infectious bronchitis virus infects the trachea and lungs of chickens, causing respiratory distress, especially in young chicks. The virus also replicates in kidneys, bursa, and oviducts, producing inflammatory disease at these sites. Human respiratory coronaviruses are apparently restricted to the upper respiratory tract, causing rhinotracheitis and pharyngitis.


References

Bhatt PN, Jacoby RO (1977) Experimental infection of adult axenic rats with Parker's rat coronavirus. Arch Virol 54: 345-352

Bhatt PN, Percy DH, Jonas AM (1972) Characterization of the virus of sialodacryoadenitis of rats: a member of the coronavirus group. J Infect Dis 126: 123-130

Bohl EH (1981) Coronaviruses: diagnosis of infections. In: Kurstak E, Kurstak C (eds) Comparative diagnosis of viral diseases, vol 4. Academic, New York, chap 7


Jonas AM, CraftJ, Black CL, Bhatt PN, Hilding D (1969) Sialodacryoadenitis in the rat. A light and electron microscopic study. Arch Pathol88: 613-622

Parker JC, Cross SS, Rowe WP (1970) Rat coronavirus (RCV): a prevalent, naturally occurring pneumotropic virus of rats. Arch Virusforsch 31: 293-302

Pneumonia Virus of Mice Infection, Lung, Mouse and Rat

David G. Brownstein

Synonyms. Mouse pneumonia virus; pneumonia VlruS.

Gross Appearance

Mice. Gross lung changes are not seen in mice naturally infected with pneumonia virus of mice (PVM). Pulmonary consolidation has been produced experimentally using tissue-culture-adapted virus (Harter and Choppin 1967; Tennant et al. 1965) or serially blind-passed PVM-infected lung tissue (Horsfall and Hahn 1940; Curnen and Horsfall 1947). Initially, consolidation is hilar in distribution with subsequent radiation along bronchioles. Consolidated foci are dark red and exude sanguinous fluid when cut. Later these foci become gray.

Rats. Naturally infected adult rats usually have no gross lung changes but may have focal or multifocal plum-colored to gray foci less than 2 mm in diameter. These may occur in any lobe.


Mice. Histopathologic lung changes are rare in naturally infected mice. Lesions produced experimentally with tissue-culture-adapted (Carthew and Sparrow 1980a; Vogtsberger et al. 1982) or serially blind-passed infected lung tissue (Horsfall and Hahn 1940) include both airway and parenchymal changes. PVM adapted to BHK-21 cells and inoculated at high doses (104-105 TCID50) in-

tranasally causes a mild erosive bronchiolitis and interstitial pneumonia. Bronchiolar epithelium first develops granular eosinophilic cytoplasm followed by lifting from the basal lamina. Desquamated epithelium and neutrophils may plug affected airways. Alveolar changes generally lag behind those in the airways. Alveolar septa are edematous, congested, and infiltrated with neutrophils and macrophages. There is some necrosis within alveolar walls. By the time bronchiolar epithelium has regenerated and returned to a normal appearance, alveolar septa are thickened by dense infiltrates oflymphocytes, macrophages, and scattered neutrophils. Alveolar spaces may contain an exudate with a similar inflammatory cell composition. Inflammatory changes in the parenchyma peak near the end of the 2nd week of experimental infection and are usually resolved by the end of the 3rd week. Lower doses « 103 TCID50) of tissue-cultureadapted PVM cause vascular-oriented inflammation and interstitial pneumonia (Vogtsberger et al. 1982). The vascular component has been described as an acute vasculitis with infiltrates of neutrophils and lymphocytes followed by a mild nonsuppurative vasculitis, which persists to the end of the 3rd week of infection. The interstitial pneumonia is acute, but its duration and histological features have not been reported. Mild rhinitis is a constant feature in mice infected intranasally with tissue-culture-adapted PVM, even at doses that fail to produce pulmonary histopathology. The nasal mucosa is edematous with multi focal erosions. Neutrophils and lymphoid cells infiltrate the lamina propria. A sparse ex-

Pneumonia Virus of Mice Infection, Lung, Mouse and Rat 207

Fig.251 (Above). Perivascular infiltrates and interstitial pneumonia in a rat naturally infected with PVM. Hand E, x 64 (reduced by 15%)

udate rich in neutrophils and desquamated epithelium is occasionally present.

Rats. Histopathologic changes in lung have been observed in naturally and experimentally infected rats. Naturally infected weanling Lewis rats develop hyperplasia of bronchus-associated lymphoid

Fig.252 (Below). Perivascular plasma cell infiltrates in a PVM-infected rat. Hand E, x 217 (reduced by 15%)

tissue (BAL T), perivascular mononuclear cell infiltrates, and multifocal interstitial pneumonia (Fig. 251). Prominent germinal centers form within the expanded BALT. The overlying airway epithelium is intact but bulges into the lumen. Transepithelial lymphocytes are increased near these reactive lymphoid nodules. Pulmonary venules,


Fig. 253. Interstitial pneumonia in a PVM-infected rat. Alveolar septa are congested, edematous, and infiltrated with mixed mononuclear cells. Alveolar lining cells are hy-

small veins, and aterioles contain dense, usually symmetrical, adventitial infiltrates of plasma cells, reactive lymphoid cells, and macrophages (Fig.252). The endothelium of these vessels may be hypertrophied and leukocyte pavementing may be prominent. Perivascular infiltrates often occur in areas of interstitial inflammation. Alveolar septa are congested, edematous, and infiltrated with lymphocytes, plasma cells, and macrophages. Alveolar lining cells are swollen or hypertrophied. Alveolar spaces contain variable numbers of foamy macrophages, desquamated pneumocytes, lymphocytes, and neutrophils (Fig. 253). Multifocal nonsuppurative vasculitis and acute interstitial pneumonia have been described in experimentally infected Fischer 344 rats (Vogtsberger et al. 1982).

Ultrastructure

The fine structure of PVM infection has only been described in cell cultures (Compans et al. 1967; Berthiaume et al. 1974). Nucleocapsid assembly occurs in the cytosol. Pleomorphic inclusions, which may be seen by light microscopy in the vicinity of the nucleus, are composed of electrondense dots or threads approximately 12 nm in di-

pertrophied. Alveolar spaces contain large foamy mononuclear cells - probably desquamated pneumocytes and macrophages. Hand E, x 536 (reduced by 15%)

ameter. Virus maturation occurs at the plasma membrane, where budding particles have round or filamentous profiles about 80-120 nm in diameter with filamentous forms predominating. The viral envelope contains surface projections approximately 12 nm in length. Within the envelope are four to eight electron-dense dots or strands identical to those seen in the cytosol. Strands are usually coiled in filamentous forms. Terminal swellings, 150-300 nm in diameter, are usually present on filamentous forms. In negatively stained preparations, filamentous forms predominate with lengths up to 3 !-Lm and diameters of approximately 100 nm.


Mice. Naturally occurring PVM apparently rarely causes pneumonia. Until there is evidence to the contrary, PVM should be considered an unlikely cause of inflammatory lung disease in mice under natural conditions, but should be regarded as a cause of mild erosive rhinitis.

Rats. The pulmonary changes in PVM infection must be distinguished from those caused by Sendai virus, rat coronaviruses, Mycoplasma pulmonis. and pathogenic bacteria. The lesions caused

Pneumonia Virus of Mice Infection, Lung, Mouse and Rat 209

by PVM most closely resemble those caused by pneumotropic strains of rat coronaviruses (Parker's rat coronavirus). Generally, the latter causes milder lesions than PVM.

Biologic Features

Natural History. Pneumonia virus of mice causes acute limited infections in immunocompetent rodents. Virus peristence (over 20 days) has been reported in germ-free athymic (nu/nu) mice (Carthew and Sparrow 1980b). Enzootic and epizootic forms exist and both are asymptomatic in mouse and rat colonies. Transmissibility in mouse colonies is low; infections may therefore be focal (Tennant et al. 1966). Attack rates in 59% of infected mouse colonies are reported to be 25% or less. This is the lowest attack rate for any indigenous murine virus (Tennant et al. 1966). Different age groups may harbor the infection, depending on management practices. In one colony, antibody was first detected in 8-week-old mice, while in a second colony, mice did not seroconvert until the age of 7 months (Parker et al. 1966). Attack rates are apparently higher in rat colonies. We frequently find a 100% prevalence of serum antibodies (hemagglutination inhibition test) in weanling rats from enzootically infected colomeso Clinical signs have not been reported for naturally infected mice or rats. Depression, anorexia, weight loss, ruffled fur, hunched posture, and labored respiration have been reported in experimentally infected mice (Horsfall and Hahn 1940).

Pathogenesis. Viral replication is restricted to the epithelium of the respiratory tract (Carthew and Sparrow 1980a, b). Infectious virus can be detected up to 10 days after exposure (A. L. Smith and V. A. Carrano personal communication). Viral antigens have not been detected in lung sections beyond day 7 in mice (Carthew and Sparrow 1980a). Virus is most consistently isolated from nasal washes in experimentally or naturally infected rodents (A. L. Smith and V. A. Carrano, personal communication). Seroconversion (hemagglutination inhibition [HAl], complement fixation [CF]) usually occurs 9 or 10 days after exposure. CF antibody titers begin to decline during the 3rd week of infection, while HAl antibody titers remain elevated for at least 4 months (Tennant et al. 1966).

Etiology. Pneumonia virus of mice is a Pneumovirus of the family Paramyxoviridae. It shares this genus with respiratory syncytial virus, which has structural and biologic but not antigenic similarities (Joncas et al. 1969; Berthiaume et al. 1974). PVM is a predominantly filamentous enveloped virus containing a single-stranded RNA genome. The hem agglutinins probably occur within the fringe of envelope projections. The virus is labile in the environment and rapidly inactivates at room temperature.

Frequency. Pneumonia virus of mice is prevalent in mouse and rat colonies throughout the world. In a recent survey, 63% of mouse and 68% of rat colonies from institutional and commercial sources were infected (Parker and Richter 1982).


Pneumonia virus of mice and respiratory syncytial virus (RSV) are the sole representatives of the genus Pneumovirus. PVM infects rodents naturally; RSV infects children, cattle, and sheep, and experimentally infects ferrets and cotton rats. Both of these viruses have potentially broad respiratory epitheliotropism that is differentially expressed depending on the host, host age, and in-, fecting dose of virus. Experimentally, PVM causes rhinitis, bronchiolitis, or interstitial pneumonia in mice. Naturally occuring PVM infection in rats causes interstitial pneumonia. RSV in humans and cattle causes rhinitis, bronchiolitis, or interstitial pneumonia (Aherne et al. 1970; Mohanty et al. 1975). Experimentally, RSV produces rhinitis and interstitial pneumonia in infant ferrets (Prince and Porter 1976) and bronchiolitis in cotton rats (Prince et al. 1978).

References

Aherne W, Bird T, Court SD, Gardner PS, McQuillin J (1970) Pathological changes in virus infections of the lower respiratory tract in children. J Clin Pathol 23: 7-18

Berthiaume L, Joncas J, Pavilanis V (1974) Comparative structure, morphogenesis and biological characteristics of the respiratory syncytial (RS) virus and the pneumonia virus of mice (PVM). Arch Virusforsch 45: 39-51

Carthew P, Sparrow S (1980a) A comparison in germ-free mice of the pathogenesis of Sendai virus and mouse pneumonia virus infections. J Pathol130: 153-158

Carthew P, Sparrow S (1980b) Persistence of pneumonia

210 David. G. Brownstein

virus of mice and Sendai virus in germ-free (nu/nu) mice. Br J Pathol61: 172-175

Compans RW, Harter DH, Choppin PW (1967) Studies on pneumonia virus of mice (PVM) in cell culture. II. Structure and morphogenesis of the virus particle. J Exp Med 126:267-276

Cumen EC, Horsfall FL Jr (1947) Properties of pneumonia virus of mice (PVM) in relation to its state. J Exp Med 85:39-53

Harter DH, Choppin PW (1967) Studies on pneumonia virus of mice (PVM) in cell culture. I. Replication in baby hamster kidney cells and properties of the virus. J Exp Med 126: 251-266

Horsfall FL, Hahn RG (1940) A latent virus in normal mice capable of producing pneumonia in its natural host. J Exp Med 71: 391-408

Joncas J, Berthiaume L, Pavilanis V (1969) The structure of the respiratory syncytial virus. Virology 38: 493-496

Mohanty SB, Ingling AL, Lillie MG (1975) Experimentally induced respiratory syncytial viral infection in calves. Am J Vet Res 36: 417-419

Parker JC, Richter CB (1982) Viral diseases of the respira-

tory system. In: FosterHL, SmallJD, FoxJG (eds) The mouse in biomedical research, vol 2, Diseases. Academic, New York, chap 8

ParkerJC, Tennant RW, Ward TG (1966) Prevalence of viruses in mouse colonies. Natl Cancer Inst Monogr 20: 25-36

Prince GA, Porter DD (1976) The pathogenesis of respiratory syncytial virus infection in infant ferrets. Am J Pathol82: 339-352

Prince GA, Jenson AB, Horswood RL, Camargo E, Chanock RM (1978) The pathogenesis of respiratory syncytial virus infection in cotton rats. Am J Pathol 93: 771-791

Tennant RW, Parker JC, Ward TG (1965) Virus studies with germ-free mice. II. Comparative responses of germ-free mice to virus infection. JNCI 34: 381-387

Tennant RW, Parker JC, Ward TG (1966) Respiratory virus infections of mice. Natl Cancer Inst Monogr 20: 93-104

Vogtsberger LM, Stromberg PC, Rice JM (1982) Histological and serological response of B6C3F1 mice and F344 rats to experimental pneumonia virus of mice infection. Lab Anim Sci 32: 419 (abstract)

Sialodacryoadenitis Virus Infection, Lung, Mouse

David G. Brownstein

Synonyms. Rat submaxillary gland virus infection; SDAV infection.

Gross Appearance

Weanling mice experimentally infected by the intranasal route develop red-brown foci distributed uniformly over all lobes of the lung (Bhatt et al. 1977).


The typical pulmonary lesion is multifocal interstitial pneumonia. This lesion is acute, with a peak intensity on postexposure day 6 and resolution beginning on days 8-10. Inflammatory foci are usually oriented around terminal bronchioles (Fig.254) and frequently radiate to the pleura. In some cases, however, orientation around terminal airways is not obvious. In these cases, inflammation is randomly distributed and usually less circumscribed than peribronchiolar inflammatory foci.

Initially, septa of affected alveoli are edematous and congested with reactive or degenerative changes in lining epithelium. Reactive cells are hypertrophied with large vesicular nuclei; degenerating cells are pyknotic and sloughing. Macrophages and, to a lesser degree, lymphocytes accumulate in the interstitium and alveolar spaces (Fig. 255). The adventitia of blood vessels adja-

Fig.254 (Above). Sialodacryoadenitis viral pneumonia in l> an experimentally infected weanling mouse. Inflammation is oriented around terminal bronchioles. Hand E, x 54 (reduced by 15%)

Fig. 255 (Middle). Sialodacryoadenitis viral infection. Early pneumonic changes in an experimentally infected mouse. Alveolar septa are congested and edematous with increased cellularity due to lymphoreticular infiltrates. At this magnification occasional swollen lining cells can be identified. Hand E, x 195 (reduced by 15%)

Fig.256 (Below). Sialodacryoadenitis viral infection. Peak pneumonic changes in an experimentally infected mouse. Alveolar septa are expanded by lymphoreticular cells and a modest mononuclear cell exudate is in the alveoli. Hand E, x 195 (reduced by 15%)

Sialodacryoadenitis Virus Infection, Lung, Mouse 211

212 David. G. Brownstein

cent to inflammatory foci contains loose eccentric aggregates of mixed mononuclear cells. Leukocyte pavementing is usually seen along the intima of these vessels. Terminal airways, around which inflammatory foci are oriented, rarely have detectable change in lining epithelium. During the period of peak inflammation, alveolar septa are markedly thickened by lymphoreticular cells (Fig. 256). Foamy macrophages, frequently in cohesive clusters, are numerous in alveolar spaces along with desquamated pneumocytes, some lymphocytes, and necrotic debris. Partial atelectasis may be present in severely inflamed foci. Resolution begins with a decline in the cellularity and thickness of alveolar septa. Foamy macrophages continue for a while to be numerous in alveolar spaces. Transient lymphoid nodules and diffuse lymphoplasmacytic infiltrates develop in the adventitia of nearby blood vessels and bronchioles. The duration of these changes is not known.

Ultrastructure

Ultramicroscopic changes have not been reported for sialodacryoadenitis virus infection in mice. The appearance of this virus in transmission electron micrographs of infected rat submaxillary glands has been reported (Jonas et al. 1969). Within infected cells, cisternae of endoplasmic reticulum and cytoplasmic vesicles contain dense or hollow spherical cores, 60-70 nm in diameter, surrounded by an envelope 40-120 nm in diameter. The corona seen in negatively stained preparations is not seen by transmission ultramicroscopy.


Sialodacryoadenitis viral pneumonia in mice must be distinguished from pneumonia caused by Sendai virus, pneumonia virus, K virus, mouse coronavirus, Mycoplasma pulmonis, and pathogenic bacteria such as Corynebacterium kutscheri. The alveolar lesion seen in SDAV infection resembles the alveolar component of Sendai virus infection but is less exudative, and is not accompanied by inflammatory airway changes. Both SDAV and certain strains of mouse coronavirus (mouse hepatitis virus) cause inflammatory lung lesions and seroconversion to mouse hepatitis virus in mouse colonies (Bhatt et al. 1977). Because

mice are apparently not natural hosts of sialodacryoadenitis virus, contact with infected rats is probably required.

Biologic Features

Natural History. This virus has not been proved to infect mice under natural conditions. Some epidemiologic evidence indicates that sialodacryoadenitis virus may account for unexpected or unexplained appearance of antibodies to mouse hepatitis virus in mouse colonies (Jacoby et al. 1979). In rats SDAV is highly contagious by aerosol, contact, and fomites. Whether mouse to mouse transmission is equally efficient has not been determined. Mice are asymptomatic during experimental SDAV infections (Bhatt et al. 1977).

Pathogenesis. This virus causes acute limited infections in experimentally inoculated mice. The virus is epitheliotropic with replication limited to the respiratory tract. It replicates at all levels of the respiratory tract but produces disease primarily in the lungs, where the highest viral titers are achieved. The tissue tropism of SDAV in mice differs from that in infected rats. In rats, virus replication and disease occur primarily in exocrine tissues of the head and epithelium of the upper respiratory tract. In mice, the principal targets are alveolar lining cells, with poor replication of virus in epithelium of the upper respiratory tract. Virus titers peak in the lungs on postexposure day 2 and are not detectable by day 8 (Bhatt et al. 1977).

Etiology. Sialodacryoadenitis virus, one of the Coronaviridae, is a pleomorphic, enveloped RNA virus with plump, pedunculated surface projections (corona). It is approximately 114nm in diameter (Jonas et al. 1969). The virus replicates intracytoplasmically and virions are formed in cytoplasmic vesicles and endoplasmic reticulum (Jacoby et al. 1979). The virus is closely related antigenically to Parker's rat coronavirus (Bhatt et al. 1972).

Frequency. Coronavirus infections are common in commercial and institutional mouse colonies. Most of these infections are due to mouse coronaviruses (mouse hepatitis virus), but the possibility that some may result from SDA V cannot be ruled out; some mice are clearly susceptible to experimental infection with the virus.


Coronaviruses are widespread in humans, animals, and birds. They produce enteritis, encephalomyelitis, sialodacryoadenitis, and systemic infections in addition to infections of the respiratory tract. Generally, respiratory coronaviruses, which infect chickens, rats, and humans, produce disease in the upper respiratory tract. There is evidence in humans, however, that coronaviruses are important causes of viral pneumonia (Mcintosh et al. 1974).

Murine Respiratory Mycoplasmosis, Lung, Rat 213

References

Bhatt PH, Percy DH, Jonas AM (1972) Characterization of the virus of sialodacryoadenitis of rats: a member of the coronavirus group. J Infect Dis 126: 123-130

Bhatt PN, Jacoby RO, Jonas AM (1977) Respiratory infection in mice with sialodacryoadenitis virus, a coronavirus of rats. Infect Immun 18: 823-827


Jonas AM, CraftJ, Black CL, Bhatt PN, Hilding D (1969) Sialodacryoadenitis in the rat. A light and electron microscopic study. Arch Pathol88: 613-622

McIntosh K, Chao RK, Krause HE, Wasil R, Mocega HE, Mufson MA (1974) Coronavirus infection in acute lower respiratory tract disease of infants. J Infect Dis 130: 502-507

Murine Respiratory Mycoplasmosis, Lung, Rat

Trenton R. Schoeb and 1. Russell Lindsay

Synonyms. Murine chronic respiratory disease; chronic murine pneumonia; enzootic bronchiectasis.

Gross Appearance

The lungs are externally normal in the majority of infected rats, gross lesions being poorly correlated with clinical signs and microscopic changes. The causative organism, Mycoplasma pulmonis, preferentially affects the nasal passages and middle ears, and the incidence of lesions decreases from the nose distally. The bronchi are the most commonly affected parts of the lungs. Purple, depressed areas of atelectasis may occur in lungs in which exudate obstructs airways. A few rats with advanced disease have gray- or yellow-purple consolidated areas of pneumonia. Yellow, slightly elevated foci representing bronchioles dilated with purulent exudate can affect entire lobes, imparting a cobblestone appearance (Figs.257 and 258). Frank abscesses also are seen in a few cases. The bronchial and paratracheallymph nodes may be enlarged to three or four times normal size.

Fig. 257. Murine respiratory mycoplasmosis, rat. Severe diffuse bronchiolectasis, atelectasis, and pneumonia in the left lung. x 2

214 Trenton R. Schoeb and 1. Russell Lindsay


The lesions of murine respiratory mycoplasmosis are so common that many pathologists lack familiarity with the histology of normal rat lungs (Figs.259 and 260), which differs slightly from that of many other species. First, pathogen-free and even germfree laboratory rats have small amounts of bronchus-associated lymphoid tissue, especially at the acute angles of bronchial bifurcations, and there are also lymphoid aggregates between the bronchi and adjacent blood vessels. Second, the trachea, bronchi, and bronchioles are lined by cuboidal to low columnar epithelium, rather than the tall columnar, pseudostratified type. Lesions in the lung usually occur mainly in the major airways and are characterized by neutro-

Fig.258. Murine respiratory mycoplasmosis, rat. Severely affected left lung with bronchiectasis, bronchiolectasis, atelectasis, pneumonia, and greatly increased bronchial lymphoid tissue. x 8

philic exudate, epithelial hyperplasia, and hypertrophy in varying degrees, and an increase in peribronchial lymphoid tissue (Figs. 261-263). The distribution and severity of these changes within the lungs, and even within an individual lobe, are quite variable. Bronchiectasis and bronchiolectasis often result as airways become distended with purulent exudate. After weeks or months, the epithelium of these distended airways becomes flattened ("squamoid") or even nonkeratinizing stratified squamous. Less commonly, the epithelium is destroyed completely, and lost in the resulting abscess. The epithelium of alveoli around these severely affected airways may become cuboidal, imparting a glandular appearance (Fig. 263). Variably distributed alveolar exudation of neutrophils and macrophages is a feature of advanced disease in which infection has spread centrifugally beyond the bronchioles.

..

Fig. 259. Normal lungs of a rat with small amounts oflymphoid tissue predominantly at major bronchial bifurcations. x 4 (reduced by 10%)

Ultrastructure

Mycoplasma pulmonis parasitizes the surface of respiratory epithelial cells. Heavy infections induce degenerative changes, such as loss of cilia and cytoplasmic vacuolation. Some cells also undergo more severe changes, indicating irreversible damage (necrosis), but it is not established whether such damage is due to the organism, to associated inflammatory processes, or both.


In naturally occurring respiratory disease in rats, M.pulmonis is almost universally present. One or more other bacteria also may be found, such as Streptococcus pneumoniae, Corynebacterium

Fig. 260. Normal bronchial lymphoid tissue of rat with flat epithelium and mostly small lymphocytes. Hand E, x 450


kutscheri, Bordetella bronchispetica, Pasteurella pneumotropica, Streptobacillus moniliformis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and an unidentified argyrophilic bacillus which also parasitizes the surface of respiratory epithelial cells (MacKenzie et al. 1981; van Zwieten et al. 1980). Most of these organisms are probably only opportunistic pathogens but S. pneumoniae and C. kutscheri are considered to be primary pathogens (Weisbroth 1979). S.pneumoniae causes fibrinopurulent bronchopneumonia, pleuritis, and pericarditis, and C. kutscheri induces multifocal suppurative pneumonia. Both organisms can be demonstrated in sections by tissue gram stains. These diseases are easily differentiated from murine respiratory mycoplasmosis, but because affected rats commonly have concurrent mycoplas-

Fig. 261. Mildly affected lungs of a rat with mild to moderate increase in bronchial lymphoid tissue and early bronchiolitis in the right cranial lobe and cranial part of the left lung. x 4

216 Trenton R. Schoeb and J. Russell Lindsay

mal disease, cultural, morphological, and serologic evidence of respiratory mycoplasmosis should still be sought. Many rats with respiratory mycoplasmosis also have concurrent Sendai virus, sialodacryoadenitis virus, or rat coronavirus infection. Sendai virus infection in adult rats is usually subclinical. Lesions are similar to those in mice and are characterized by necrotizing bronchiolitis (Jacoby et al. 1979). Sialodacryoadenitis and rat coronavirus have been reported to cause multifocal interstitial pneumonia in natural or experimental infections, but these agents to no appear to be important respiratory pathogens for rats. None of these lesions, if found, should be difficult to distinguish from those of mycoplasmosis. An accurate diagnosis requires diligent efforts to

Fig.262. Bronchus in murine respiratory mycoplasmosis with neutrophilic exudate, mild hyperplasia of respiratory epithelium, intraepitheliallymphocytes, and large lymphocytes and plasmacytoid cells in the lamina propria. Hand E, x 450

determine which viruses and bacteria are present, using several methods including microscopic examination of tissues, bacterial and mycoplasmal culturing, and serologic testing. One must critically evaluate and correlate all results to make diagnoses appropriate to each animal or colony. Failure to isolate M. pulmonis is not evidence of its absence, inasmuch as there are many difficulties in isolating this organism by ordinary methods. For example, certain tissue substances and even medium components can be inhibitory (Del Giudice et al. 1980; Kaklamanis et al. 1971; Mardh and Taylor-Robinson 1973; Tauraso 1967). Culturing several sites in the respiratory tract is advantageous, as is combining culture with other diagnostic methods (Davidson et al. 1981), such as enzyme-linked immunsorbent assay (Horowitz

• • •

"PI • • . ,:,'."- .~ ~ I . •• ., ' • . . ;

.to. ~ •

Fig.263. Bronchiole in severe murine respiratory mycoplasmosis with neutrophilic exudate, severe epithelial distortion and ciliary loss, lymphoid accumulation, and peribronchiolar glandular structures. Hand E, x 450

and Cassell 1978), which is now available commercially. Although microbial agents present simultaneously with M. pulmonis probably modify the expression of natural respiratory disease in rats, no other agent has been demonstrated to produce lesions resembling those of mycoplasmosis in rats that are clearly free of other pathogens. Therefore, M. pulmonis should be considered the primary pathogen in rats having lesions consistent with those of experimental murine respiratory mycoplasmosis.

Biologic Features

The biologic features are discussed further on page 80. The roles of the various specific and nonspecific host defense mechanisms in resistance of rats to mycoplasmosis are unclear. Systemic antibody and cellular responses occur, but the cellular response appears more important in rats inasmuch as resistance can be conferred by transfer of cells but not serum (Cassell et al. 1973). Vaccination studies have shown that resistance to disease can be induced by local or systemic vaccination, and have provided circumstantial evidence for the existence of both local and systemic responses (Cassell and Davis 1978). A vigorous local response would seem to be indicated by the charateristic lymphoid accumulations; however, much of this could be due to nonspecific mitogenic activity of M. pulmonis (N aot et al. 1979). It remains to be determined whether the lymphoid accumulation is due to infiltration, local proliferation, or both. In vivo studies have indicated that alveolar macrophages may be important in resistance to alveolar invasion by M.pulmonis (Cassell et al. 1973). However, in vitro experiments have demonstrated only inhibition of multiplication of M.pulmonis by alveolar macrophages rather than rapid killing of the organism (Davis et al. 1980). Like other respiratory mycoplasmas, M. pulmonis parasitizes the surface of ciliated epithelial cells. The mechanisms by which it affects these cells are unclear, but possibilities include competition for metabolites or components of the host cells and production of toxic wastes (Cassell et al. 1978). The close association with host cells may contribute to the ability of M.pulmonisto escape elimination by host defenses (Cassell et al. 1978). For example, it may prevent mucociliary clearance, phagocytosis, or efficient attack by specific immune effector mechanisms. However, alteration of lymphocyte responsiveness and consequent


misdirection or disruption of immune responses by nonspecific mitogenicity (Naot et al. 1979) also seem likely contributors to the organism's virulence, ability to resist elimination, or both (Cassell et al. 1979). Mycoplasma pulmonis infection is ubiquitous in conventional rat colonies, and has been identified in "barrier-maintained" colonies in the United States and Great Britain by serologic testing and cultural isolation (Cassell et al. 1981). It also has been found in colonies thought to be germ free (Ganaway et al. 1973).


Comparative aspects of mycoplasmosis in several species are discussed on page 82.

References

Cassell GH, Davis JK (1978) Protective effect of vaccination against Mycoplasma pulmonis respiratory disease in rats. Infect Immun 21: 69-75

Cassell GH, Lindsey JR, Overcash RG, Baker HJ (1973) Murine mycoplasma respiratory disease. Ann NY Acad Sci 225: 395-412

Cassell GH, Davis JK, Wilborn WH, Wise KS (1978) Pathobiology of mycoplasmas. In: Schlessinger D (ed) Microbiology 1978. American Society for Microbiology, Washington DC, pp 399-403

Cassell GH, Lindsey JR, Baker HJ, Davis JK (1979) Mycoplasmal and rickettsial diseases. In: Baker HJ, Lindsey JR, Weisbroth SH (eds) The laboratory rat, vol!. Academic, New York, chap 10

Cassell GH, Lindsey JR, Davis JK, Davidson MK, Brown MB, Mayo JG (1981) Detection of natural Mycoplasma pulmonis infection in rats and mice by an enzyme linked immunosorbent assay (ELISA). Lab Anim Sci 31:676-682

Davidson MK, Lindsey JR, Brown MB, Schoeb TR, Cassell GH (1981) Comparison of methods for detection of Mycoplasma pulmonis in experimentally and naturally infected rats. J Clin Microbiol14: 646-655

Davis JK, Delozier KM, Asa DK, Minion FC, Cassell GH (1980) Interactions between murine alveolar macrophages and Mycoplasma pulmonis in vitro. Infect Immun 29: 590-599

Del Giudice RA, Gardella RS, Hopps HE (1980) Cultivation of formerly noncultivable strains of Mycoplasma hyorhinis. CUff Microbiol4: 75-80

Ganaway JR, Allen AM, Moore TD, Bohner HJ (1973) Natural infection of germ-free rats with Mycoplasma pulmonis. J Infect Dis 127: 529-537

Horowitz SA, Cassell GH (1978) Detection of antibodies to Mycoplasma pulmonis by an enzyme linked immunosorbent assay. Infect Immun 22: 161-170


218 1. K. Frenkel

Kaklamanis E, Stavropoulos K, Thomas L (1971) The mycoplasmacidal action of homogenates of normal tissues. In: MadoffS (ed) Mycoplasma and the L-forms ofbacteria. Gordon and Breach, New York, pp27-35

MacKenzie WF, Magill LS, Hulse M (1981) A filamentous bacterium associated with respiratory disease in wild rats. Vet Pathol18: 836-839

Mardh PA, Taylor-Robinson D (1973) New approaches to the isolation of mycoplasmas. Med Mikrobiol Immunol (Beri) 158: 259-266

Naot Y, Merchav S, Ben-David E, Ginsburg H (1979) Mitogenic activity of Mycoplasma pulmonis. I. Stimu-

Pneumocystosis, Lung, Rat

J. K. Frenkel

Synonym. Interstitial plasma cell pneumonia.

Gross Appearance

Heavily infected lungs are focally to diffusely consolidated but never involve an entire lobe. The alveoli are filled with grayish material, between which the pinkish alveolar walls can be seen with a hand lens (Fig. 264). If tissue necrosis or abscesses are present, another organism should also

Fig.264. Pneumocytosis, lung of rat. The dorsal aspect of both lungs is diffusely pale pink opaque with only few alveoli containing trapped air (light, refractile areas). Rat

lation of rat Band T lymphocytes. Immunology 36: 399-406

Tauraso NM (1967) Effect of diethylaminoethyl dextran on the growth of mycoplasma in agar. J Bacteriol 93: 1559-1564

van Zwieten MJ, Solleveld HA, Lindsey JR, de Groot FG, Zurcher C, Hollander CF (1980) Respiratory disease in rats associated with a filamentous bacterium: a preliminary report. Lab Anim Sci 30: 215-221

Weisbroth SH (1979) Bacterial and mycotic diseases. In: Baker HJ, Lindsey JR, Weisbroth SH (eds) The laboratory rat, vol 1. Academic, New York, chap 9

be looked for. In contrast to consolidated lungs, the lungs are not hyperemic and are less voluminous. No fibrinous pleuritis occurs unless bacterial or fungal pneumonia is present.


The alveoli are filled with masses of Pneumocystsis organisms, usually with a few accompanying macrophages (Figs. 265 and 266). In hematoxylin-

was treated with 25 mg cortisone acetate twice weekly and with 1 mg amphotericin subcutaneously three times weekly for 75 days (reduced by 30%)

and eosin-stained sections the intraalveolar masses appear regularly vacuolated, comparable to a honeycomb (Fig. 266). With the periodic acidSchiff technique the honeycombed material is more intensely stained; hematoxylin-stained nuclei are less distinct (Fig.267). Using the Grocott modification of Gomori's methenamine silver technique, black yeast-like cysts measuring 3-5 11m in diameter are found individually or in groups in some of the alveolar masses (Fig. 268). The number of these cysts is variable: it is less than the number of nuclei staining with hematoxylin and many alveoli with honeycombed masses contain few or no cysts. The silver technique generally demonstrates only the cyst wall, not the enclosed nuclei (Fig. 269). The cysts are best identified in Giemsa-stained imprints, where they appear as clear, round spaces against a dark-staining protein background with eight dark-staining nuclei. Methyl violet, toluidine blue, and related dyes also stain the cyst walls. Several rapid staining techniques have been described (see, for example, Macher et al. 1983).

Ultrastructure

Trophozoites are ameboid in shape and measure 1-5 11m with a 20-30 nm pellicle, apparently composed of a double layer. Filopodia extend from the surfaces. The nucleus is bordered by a singleor double-layered membrane which is poorly defined. The cytoplasm contains mitochondria, ribosomes, endoplasmic reticulum, some vacuoles, lipid globules, and granules, some of which stain for glycogen (Campbell 1972). A precyst, limited by a semirigid trilayered membrane, contains one to several masses of nucleoplasm. Cysts measure 3.5-5 11m in diameter and are also limited by a trilayered wall, 50-300 nm in thickness. An inner unit type membrane is 5-7 nm thick. A middle granular electrolucent layer usually measures 30-40 nm in diameter, with occasional thicker portions. The outer layer is granular, electron dense, and 20-75 nm in thickness. It contains several nuclear masses or separate intracystic bodies, mitochondria, glycogen, and vacuoles. Filopodia may be attached to the surface (Campbell 1972). Intracystic bodies measure 1-2 11m in diameter and are enclosed by a double-layered membrane 20-30 nm thick. Their nuclei measure less than 111m. The nuclear envelope is sometimes continuous with the endoplasmic reticulum and ribosomes are often attached to the external surface.

Pneumocystosis, Lung, Rat 219

Intracystic bodies develop into extracystic trophoblasts. Empty cysts are irregular in shape and often appear crescentic because of collapse of the wall.


Since the intraalveolar accumulations of Pneumocystis appear honeycombed and contain trophozoite nuclei, this lesion can be easily differentiated from pulmonary edema, which often accompanies it and may be the primary finding after inhalation of toxic substances. Depending on the immune defect present, a small number of inflammatory cells, usually macrophages, may be found. Alveolar wall infiltration is usually slight; however, in the presence of associated bacterial or fungal infection it may be severe. Pneumocystis must be differentiated from other organisms. Their nuclei stain with hematoxylin, but are less densely stained than most gram-positive bacteria, which also stain with hematoxylin. Cyst walls stained with silver, methyl violet, or toluidine blue must be differentiated from fungi, especially Candida (Tornlopsis) glabrata (Fig. 270). The finding of crescentic or cup-shaped cysts is characteristic of Pneumocystis, as is the irregular wrinkling of the intact cyst. Budding would be characteristic for yeast; Candida glabrata, which is the most common source of confusion, can be cultured. The eight-nucleated cyst should always be searched for in Giemsa-stained imprints for confirmation of the diagnosis (Fig. 271).

Biologic Features

Natural History. Most colonies of rats are naturally infected, as are some colonies of mice and rabbits. Normally infections are subclinical and iatrogenic immunosuppression, usually with corticosteroids or cyclophosphamide, or profound malnutrition are necessary to give rise to clinically significant pulmonary involvement (Frenkel et al. 1966; Frenkel 1976; Robbins et al. 1976). Athymic nude mice are also susceptible (Walzer and Rutledge 1980). Spontaneous instances of pneumocystosis have been described in cats, horses, goats, pigs, dogs, hares, owl monkeys, chimpanzees, and others (Chandler et al. 1976). The presence of significant pulmonary involvement with Pneumocystis is an indication of immunosuppression. Hughes (1982) showed that

220 1. K. Frenkel

naturally infected lung tissue is not infectious and that rats become infected from contaminated air. The infectious stage has not yet been determined.

Pathogenesis. In normal rats pneumocystis organisms are quite scarce and the Delanoes, who described and named Pneumocystis, reported searching three or four impression smears before finding a single cyst in normal rats (Delanoe and Delanoe 1912). In athymic rats, small focal infiltrates may be found. Only after cortisone became available was massive pulmonary involvement produced in rats (Frenkel et al. 1966; Chandler et al. 1979). Earlier workers encountered Pneumocystis accompanied by pneumonia and abscesses from Corynebacterium kutscheri, staphylococci, Pseudomonas, Proteus, Streptobacillus moniliformis, Pasteurella pneumotropica, coliforms, and fungi. The use of chlortetracycline in drinking water (50 mg/dl) and of amphotericin B, 1 mg three times weekly subcutaneously, prevented most bacterial and fungal infections and permitted the study of relatively pure Pneumocystis infection. The following other regimes have led to clinical pneumocystosis in 200 g, 6-8 week old rats: 25 mg cortisone acetate given subcutaneously twice weekly alternating with cortisol; dexamethasone sodium phosphate, 1 mg/l in drinking water; placing 250 g rats on 8% protein diet; cyclophos-

<I Fig.265 (Upper left). Rat lung. Most alveoli filled with masses of Pneumocystis carinii (P); trapped air in some alveoli (A). The bronchi (B) contain a small amount of fluid, devoid of Pneumocystis. PAS and H, x 80

Fig.266 (Upper right). Alveolar filling with Pneumocystis. Rat was treated with 25 mg cortisone acetate twice weekly, subcutaneously, for 64 days. With hematoxylin and eosin, the organisms appear as faintly hematoxylin-staining nuclei in a foamy eosinophilic material. A few inflammatory macrophages are present. Hand E, x 630

Fig.267 (Lower left). Same lung as in Fig. 266. With the PAS-hematoxylin technique, the foamy matrix is emphasized, but the hematoxylin-staining nuclei can also be seen. PAS and H, x 630

Fig.268 (Lower right). Same lung as in Fig.266.With the Grocott methenamine silver technique, the cysts resemble spherical or collapsed yeasts, such as Histoplasma or Torulopsis;this technique is useful mainly to identify candidate forms which must be confirmed by Giemsa staining in smears; some collagen and reticulum fibers are also present. Grocott methenamine silver, x 630


Fig.269. Pneumocystis carinii cysts in section of lung impregnated with silver appear as flattened, wrinkled, or folded yeast-like bodies. Grocott methenamine silver, x 1500

phamide, 240 mg/kg body weight orally three times weeky; aminopterin and chlorambucil together with subeffective doses of cortisone acetate; and cyclosporin A 10 mg/kg per day orally. Masses of Pneumocystis tachyzoites fill the alveoli, leading to anoxia in immunosuppressed hosts (Fig. 266). Ordinarily, little inflammatory reaction is present, in part because the organism is not highly chemotactic and, in part, because the immunosuppressive state is accompanied by an impaired inflammatory response. Inflammation appears when immunocompetence returns, for example when corticosteroid administration is stopped. Inflammation also appears when the organisms are killed by the administration of pentamidine, or by sulfonamide and a dihydrofolate reductase inhibitor (Frenkel et al. 1966; Hughes et al. 1974), even though corticosteroids are still administered. All of these causes of resurgent inflammatory reaction may initially result in further

222 1. K. Frenkel

<1 Fig. 270 (Above). Fungal yeasts. This strain (of Candida (Toru/opsis) glabrata) has ovoid singular nuclei and would not easily be confused with Pneumocystis, but round yeasts could be easily confused. Smear from culture. Giemsa, x 1500

Fig.271 (Below). A Pneumocystis cyst in impression smear with eight-nucleated interior bodies (center). Numerous Pneumocystis trophozoites in the periphery. A distinct nucleus and cytoplasm can be seen. Giemsa, x 1500

impaired alveolocapillary diffusion and increased anoxia, until some of the Pneumocystis are eliminated. The organisms are essentially confined to the alveoli, where they are closely attached to type I pneumocytes, which later degenerate (Walzer et al. 1980; Yoneda and Walzer 1981). They are sometimes phagocytized and may be carried to hilar lymph nodes, but have not been reported to multiply there in laboratory animals. Because of their tight attachment to type I pneumocytes, the organisms are not expectorated and are not found in the bronchi, except after the administration of chemotherapy with sulfadiazine-pyrimethamine, sulfamethoxazole-trimethoprim, or pentamidine.

Etiology. Carlos Chagas first recognized the eightnucleated cysts in lungs of guinea pigs infected with Trypanosoma entzi and, believing it to indicate schizogony and to be part of the trypanosomal cycle, created the genus Schizotrypanum (Chagas 1909). Pneumocystis carinii was described as a separate organism by Delanoe and Delanoe (1912) in the lungs of rats and later in guinea pigs from Paris. The organism had generally been regarded as a protozoan, although in the absence of characteristic organelles, its affinity to protozoan orders remains obscure. On the basis of the study of its ultrastructure, Vavra and Kucera (1970) stressed the similarity of this organism to fungi. Although the cyst resembles a fungus, the ameboid trophozoites do not appear to, being furthermore devoid of chitin, although other polysaccharides are present. The susceptibility of Pneumocystis to pentamidine is shared by two protozoa, Leishmania and Trypanosoma, and by the fungus Histoplasma; however, the resistance of Pneumocystis to amphotericin B makes a fungal classification less likely, although some fungi are resistant. It is likely that the taxonomic affinities of the agent will become clearer once the infectious stage has been identified.

Frequency. All conventional rats examined in 1964 and 1965 and even five or six lines advertised as "specific-pathogen-free" (SPF) developed pneumocystosis when treated with cortisone acetate, 25 mg twice weekly: one SPF line and one exgerm-free line, recently contaminated with bacteria, remained free of Pneumocystis at the same time.


Pneumocystosis in man was recognized in institutionalized premature babies and in infants at the end of World War II in Europe. The histologic picture was of an interstitial plasma cell pneumonia with masses of Pneumocystis filling the alveoli. Plasma cell pneumonia has not been observed in experimental animals. In an immunosuppressed baby with congenital rubella, both plasma cells and large mononuclear cells are present. However, no significant interstitial inflammation, as in the experimental models, is found in children with agammaglobulinemia due to deficiency of B cells (Bruton's disease), in patients with leukemia or lymphoma treated with appropriate chemotherapy, or in organ transplant recipients immunosuppressed with corticosteroids and azathioprine. Because there are serological differences between P. carinii of rats, the human organism was designated Pneumocystis jiroveci (Frenkel 1976). Athymic nude mice are sometimes observed with focal Pneumocystis accumulations; cortisonetreated rabbits and mice are also found to be infected (Sheldon 1959; Walzer et al. 1980). Recently, pneumocystosis has been described in humans with the acquired immunodeficiency syndrome and in nonhuman primates which were immunosuppressed for unknown reasons (Chandler et al. 1976).


References

Campbell WG JR (1972) Ultrastructure of Pneumocystis in human lung. Arch Pathol93: 312-324

Chagas C (1909) Nova tripanozomiaza humana. Estudos sobre a morfolojia e 0 cicio evolutivo de Schizotrypanum crnzi n.gen., n. sp. ajenta etiologio de nova entidade morbida de homan. Mem Inst Oswaldo Cruz 1 : 159-164

Chandler FW, McClure HM, Campbell WG Jr, Watts JC (1976) Pulmonary pneumocystosis in nonhuman primates. Arch Pathol Lab Med 100: 163-167

Chandler FW Jr, FrenkeUK, Campbell WG Jr (1979) Pneumocystis pneumonia. Animal model: Pneumocystis carinii pneumonia in the immunosuppressed rat. Am J PathoI95:571-574

De1anoe P, Delanoe Mme (1912) Sur les rapports des kystes de Carini du poumon des rats avec Ie Trypanosoma lewisii. C. R. Sequces Acad Sci 155: 658-664

FrenkeUK (1976) Pneumocystis jiroveci n. sp. from man: morphology, physiology, and immunology in relation to pathology. Natl Cancer Inst Monogr 43: 13-30

Frenkel JK, Good JT, Shultz JA (1966) Latent Pneumocystis infection of rats, relapse and chemotherapy. Lab Invest 15: 1559-1577

Hughes WT (1982) Natural mode of acquisition for de novo infection with Pneumocystis carinii. J Infect Dis 145: 842-848

Hughes WT, McNabb OC, Makres TD, Feldman S (1974) Efficacy of trimethoprim and sulfamethoxazole in the prevention and treatment of Pneumocystis carinii pneumonitis. Antimicrob Agents Chern other 5: 289-293

Macher A, Shelhamer J, Parker M, Parrillo J, Gill V, MasurH (1983) Emergency open lung biopsy at NIH: use of a new modified methylene blue stain for the rapid tissue demonstration of Legionella, Pneumocystis, cytomegalovirus, Nocardia and fungi causing opportunistic pneumonias. Crit Care Med 11: 221 (abstract)

RobbinsJB, DeVitaVT Jr, DutzW (eds) (1976) Symposium on Pneumocystis cariniiinfection. Natl Cancer Inst Monogr43

Sheldon WH (1959) Experimental pulmonary Pneumocystis carinii infection in rabbits. J Exp Med 110: 147-160

VavraJ, Kucera K (1970) Pneumocystis carinii delanoe, its ultrastructure and ultrastructural affinities. J Protozool 17: 463-483

Walzer PD, Rutledge ME (1980) Comparison of rat, mouse, and human Pneumocystis carinii by immunofluorescence. J Infect Dis 142: 449

Walzer PD, Powell RD Jr, Yoneda K, Rutledge ME, Milder JE (1980) Growth characteristics and pathogenesis of experimental Pneumocystis carinii pneumonia. Infect Immun 27: 928-937

Yoneda K, Walzer PD (1981) Mechanism of pulmonary alveolar injury in experimental Pneumocystis carinii pneumonia in the rat. Br J Exp Pathol62: 339-346

224 1. K. Frenkel

Aspergillosis and Mucormycosis, Lung, Rat

J. K. Frenkel

Synonyms. None (aspergillosis); phycomycosis, zygomycosis, hyphomycosis (mucormycosis).

Gross Appearance

Members of fungal species give rise to focal and diffuse pneumonia, which often progresses to necrosis, vascular invasion and infarction in immunosuppressed rats, and occasionally in mice and hamsters. The affected areas are raised with white, necrotic centers which are surrounded by gray, consolidated tissue and hyperemic borders (Fig. 272). Fibrinous pleuritis usually accompanies the lesions.


Diffuse pneumonia is accompanied by fungal hyphae, generally with tissue necrosis and infarc-

Fig. 272. Rat lung with two white plaques (arrows) covering the pleural surface. The rat was treated with 25 mg cortisone acetate subcutaneously, twice weekly for 64 days

tion. The fungi may exhibit nonseptate hyphae, characteristic of Mucor, or septate hyphae, compatible with Aspergillus. Fungal bronchitis is common with Candida, a fungal yeast, but Aspergillus and Mucor also give rise to bronchitis. Aspergillus infection next to a bronchus may erode into it, forming an ulcer, a cavity, and sometimes a fungal ball. Growth of the fungus may occur through the pleura into the pleural sac (Figs. 273 and 274). The cellular exudate contains mononuclear cells and granulocytes, which occasionally leads to granuloma formation. Aspergillus hyphae are of approximately constant width, are septate, and often undergo dichotomous branching. In the presence of a bronchus or a cavity, conidia (fruiting bodies) may be formed. The agent of Mucor has hyphae of greatly varied width which are sparsely septate with haphazard branching; the hyphae sometimes appear twisted or folded, mistakenly suggesting septation (Fig. 275). Vascular invasion is common and blood vessels often serve as conduits for the spread of the fungi.

Ultrastructure

The ultrastructure is rarely examined because the organisms are readily visible by light microscopy and their identification depends on culture. The fungal hyphae appear as tubules with prominent cell walls.


Fungal granulomas, abscesses, and pneumonias must be differentiated from bacterial abscesses and pneumonias using impression smears, histologic sections, and cultures. Fungal and bacterial infections may both be present, sometimes even with viral infections and Pneumocystis, a protozoan, in immunosuppressed animals. Because fungal contaminants are so common, one cannot rely on culture alone, and the isolated organisms must be shown by evidence in histologic sections to be related to the lesion.

Biologic Features

Natural History. The fungi are widely distributed in the environment, especially in moist feed and bedding (Emmons 1962), and the spores are readily inhaled by animals. Immunocompetent animals control the infection, which remains subclinical; occasionally, a single aspergilloma may be found, sometimes with cavitation. Not so in the immunosuppressed animal, where focal fungal bronchitis leads to necrotizing bronchopneumonia, vascular invasion, dissemination in the lung and occasionally to the other organs, and extension to the pleura. Thrombi form in the infected vessels and may give rise to infarcts without fungi, which are mainly aerobic.

Pathogenesis. In both aspergillosis and mucormycosis, tissue invasion from the bronchi and vascular invasion are predominant. It is likely that proteolytic enzymes favor the fungi's penetration through the connective tissue; collagenases and elastases have been isolated from some of the Mucorales. Acidosis markedly lowers natural resistance of hosts to mucormycosis, as has been observed clinically in humans, usually those with diabetes. This was studied in rabbits that had received injections of alloxan (Sheldon and Bauer 1962). Acidosis temporarily decreased the levels of an inhibitory serum factor. Athymic mice appear to be resistant (Corbel and Eades 1977), but corticosteroids markedly depress the capacity to react to these fungi (Frenkel 1962). Endotoxin, hemolysins, and other factors from these fungi have been described but do not appear to play an obvious role in the pathogenesis of clinical illness. Aflatoxin, however, causes acute liver necrosis and scarring in many animals, the effective dose varying according to species; it is also a potent carcinogen, again with wide variations in sensitivity according to species and according to hormonal states (Goodall and Butler 1969).

Etiology. Several species of Aspergillus (A.jlavus, A. niger, A.fumigatus) and species of Mucor, Absidia, Rhizopus, and Mortierella may be found in laboratory animals. The diagnosis is often difficult because some of the Mucorales do not grow easily in culture. Experimental infections with A.fumigatus, A.jlavus, A. niger, and Rhizopus oryzae are commonly fatal.

Frequency. Infections are rare in normal animals unless they are exposed to very large doses of fungi, as from moist and moldy feed. The frequency

Aspergillosis and Mucormycosis, Lung, Rat 225

Fig.273 (Above). Lung with fungal hyphae growing from parenchyma into pleura (F). Some of the nearby blood vessels contain similar fungi, but the bronchi are free. PAS and H, x 25

Fig.274 (Below). Aspergillus terreus (identified by culture) in pleura of rat. Septate fungal hyphae of constant bore and with occasional spores in pleura (P) and subpleural area. PAS and H, x 400

226 J. K. Frenkel

Fig.275. Mucormycosis, lung, rat. Nonseptate fungal hyphae of irregular diameter. Blood vessels were also involved, turning this area of lung into a hemorrhagic infarct. This fungus was not cultured. PAS and H, x 400

of illness is directly related to the degree of immunosuppression. The infection can be suppressed by the administration of amphotericin B, 6 mg/kg body weight (rat) administered intramuscularly three times weekly (Frenkel et al. 1966).


The tissue lesions described are quite similar in rats and humans. Mice and hamsters appear more resistant than rats. Aspergillus bronchitis is common in certain birds, leading often to disseminated infection. Allergic bronchitis due to fungi is rare in experimental animals but does occur in man. Mucorales have been reported to give rise to illness in animals fed moist and moldy food, with generalized infection common at the time of death (Jones and Hunt 1983). The rhino facial mucormycosis of humans has also been seen in a rhesus monkey (Martin et al. 1969), but appears to be rare in small laboratory animals.

References

Corbel MJ, Eades SM (1977) Experimental mucormycosis in congenitally athymic (nude) mice. Mycopathologia 62:117-120

Emmons CW (1962) Natural occurrence of opportunistic fungi. Lab Invest 11: 1026-1032

Frenkel JK (1962) Role of corticosteroids as predisposing factors in fungal diseases. Lab Invest 11: 1192-1208

Frenke1JK, Good JT, Shultz J A (1966) Latent Pneumocystis infection of rats, relapse and chemotherapy. Lab Invest 15: 1559-1577

Goodall CM, Butler WH (1969) Aflatoxin carcinogenesis: inhibition of liver cancer induction in hypophysectomized rats. Int J Cancer 4: 422-429

Jones TC, Hunt RD (1983) Veterinary pathology, 5th edn. Lea and Febiger, Philadelphia

Martin JE, Kroe DJ, Bostrom RE, Johnson DJ, Whitney RA Jr (1969) Rhino-orbital phycomycosis in a rhesus monkey (Macaca mulatta). J Am Vet Med Assoc 155:1253-1257

Sheldon WH, Bauer H (1962) The role of predisposing factors in experimental fungus infections. Lab Invest 11: 1184-1191

Toxoplasmosis, Lung, Mouse, and Hamster 227

Toxoplasmosis, Lung, Mouse and Hamster

J. K. Frenkel

Synonyms. None.

Gross Appearance

Focal to confluent pneumonias, often with serous pleural exudate, are seen at necropsy of animals which succumb to acute infection. In immunosuppressed animals in which a chronic infection relapses, white to yellowish necrotic foci surrounded by hemorrhage are seen in the lungs in addition to diffuse pneumonia.


Toxoplasma tachyzoites, the rapidly multiplying forms, are found in the alveolar walls, in cells of alveolar exudate, and in many other cells of the body (Fig. 27 6). Diffuse interstitial pneumonia is the usual picture. More rarely, focal necrosis involves segments of the alveolar wall. Intracellular and free tachyzoites, a mixed mononuclear cell infiltration, and serofibrinous exudate accompany both (Fig.277). In immunosuppressed animals, larger focal lesions involving several alveoli many be found, with diffuse cellular infiltration and necrosis or early organization by fibroblasts (Fig. 278). Focal fibrinous pleuritis is present with parenchymal lesions reaching the pleura. Rarely, Toxoplasma cysts containing bradyzoites or slowly multiplying forms are found in the lungs without inflammatory reaction.

Ultrastructure

The ultrastructure of Toxoplasma is useful to distinguish it from some other small organisms that may be encountered in the lungs. Toxoplasma tachyzoites are banana-shaped to ovoid and carry an apical complex at the anterior end composed of a conoid, a polar ring, and rhoptries, which contain enzymes that apparently aid their active penetration of host cells. Also, a vesicular nucleus, mitochondria, endoplasmic reticulum, microtubules, and micronemes are enclosed in a pellicle (Chobotar and Scholtyseck 1982).


The apical complex is present in other Sporozoa, such as intestinal and cyst-forming coccidia, which has led to a redefinition of the phylum as the Apicomplexa. Members of this group need to be differentiated by their cycle and cyst structure. However, because of their small size they must also be distinguished from Leishmania spp. and Trypanosoma cruzi, which have a kinetoplast; from Encephalitozoon, which contains a spiral polar filament; from Pneumocystis, which lacks of a special organelle; and from Histoplasma and other fungal yeasts with their PAS-positive cell wall.

Biologic Features

Natural History. Toxoplasma gondiiis an intestinal coccidian of cats with an enteroepithelial cycle that leads to the development of oocysts that are shed in the feces. Oocysts sporulate outside the host. Ground-feeding animals and herbivores become infected by ingesting vegetation mixed with soil contaminated with cat feces. These animals constitute the intermediate host; all mammals and birds investigated have been found to be susceptible. During acute infection tachyzoites multiply actively, giving rise to necrosis of infected cells. Coincident with the development of immunity, bradyzoites slowly develop within tissue cysts, with cells of the brain, muscle and other organs supporting large numbers of organisms. The cysts appear in the chronic, persisting stage of infection, and when eaten by a carnivore, produce tachyzoites. Only in felines will they initiate an intraepithelial and a sexual cycle in the gut. Laboratory animals are likely to become infected by the ingestion of oocysts from either contaminated food or bedding. Cats are known to cover their feces and will defecate sometimes in open sacks or bins containing feed and cage bedding (Fig. 279).

Pathogenesis. The tachyzoites of acute infection multiply by successive division (endodiogeny), reaching eight to 32 organisms per cell, at which time the cell usually disintegrates and the tachyzoites infect other cells. Bradyzoites develop in mice 4-5 days after infection with tachyzoites, 7-9 days

228 J. K. Frenkel

Fig.276 (Upper left). Focal toxoplasmic pneumonia, hamster (Fig.278) with tachyzoites in parenchyma, bronchial epithelium, and exudate. Groups of tachyzoites (arrows). Hand E, x 450

Fig.277 (Upper right). Toxoplasmic pneumonia. Nude mouse with partially functioning thymic transplant, died 22 days after subcutaneous inoculation with Toxoplasma tachyzoites. Group oftachyzoites (arrow). Hand E, x 400

Fig.278 (Below). Focal toxoplasmic pneumonia. Syrian golden hamster latently infected with Toxoplasma for several months. Immunosuppression with subcutaneously administered cortisol, 2.5 mg twice weekly, was then begun. Forty-five days later this animal died with focal pneumonia and focal encephalitis. Hand E, x 25

Toxoplasmosis, Lung, Mouse, and Hamster 229

HAY GRAIN

FECAL CONTAM.

MEAT ••••••••

'-"

Fig. 279. Transmission of toxoplasmosis in zoos and animal colonies.

after infection with bradyzoites, and 9-10 days after infection with sporozoites from occysts. The slow multiplication of bradyzoites leads to accumulations of tens to hundreds of organisms within intracellular cysts. These cysts contain organisms in resting stages, awaiting ingestion by a carnivore, and have little pathogenicity to the host cell, which maintains the cyst for months to years. However, disintegration of a cyst may lead to tissue necrosis in the presence of delayed hypersensitivity (type IV); this is of clinical importance when cyst rupture occurs in the retina, because function is concentrated in a few cells which do not regenerate. The liberated organisms are usually destroyed by immune mechanisms. Minimal pathogenicity is also associated with the proliferation of stages A, B, C, D, and E and the development of gametocytes in the intestinal epithelial cells of cats, because it coincides more or less with the natural turnover of epithelial cells which mature while migrating from the crypts to the tips of the villi where they are shed.

Etiology. Toxoplasma gondii was originally described as a Leishmania or related organism in 1908, both in North Africa and South America. Nicolle and Manceaux found it in gondis, wild rodents used in studies of Leishmania and typhus

at the Pasteur Institute in Tunis. These animals presumably became infected in the laboratory. Splendore found Toxoplasma in laboratory rabbits in San Paulo, Brazil. The absence of a kinetoplast separated it from Leishmania. In 1909 it was named Toxoplasma gondii by Nicolle and Manceaux (reviewed by Frenkel 1973). The organism was suspected to give rise to human disease sporadically, but it was not definitely linked with human disease until Wolf, Cowan, and Paige, starting in 1937, identified Toxoplasma in newborn babies in New York, isolated it by 1940, and linked it to intrauterine infection from an asymptomatic mother. At first the organism was called Encephalitozoon, as in a case described by lanku in 1923 (Frenkel 1973). But by 1940, when the organism had been isolated from an infected baby, it was recognized by Sabin as identical with Toxoplasma isolated from animals (Frenkel 1973). This was followed by characterization of the nednatal infection by Sabin, the development of serologic tests, especially the dye test by Sabin and Feldman, and the recognition of ocular, encephalitic, and lymph node syndromes, apart from generalized infection in children and adults. Toxoplasma was found to give rise to disease in many domestic, wild, zoo, and laboratory animals, without, because of its size and number, causing serious er-

230 J. K. Frenkel

rors in the interpretation of laboratory experiments (Frenkel 1973). Although it was realized that carnivorism could b~ responsible for transmitting toxoplasmosis, it did not become clear how herbivores became infected till 1965, when W. Hutchison discovered the infectivity of the feces of a cat that had eaten Toxoplasma-infected mice. In 1967 he described the supposed transmission in nematode eggs. Between 1969 and 1970 transmission without the nematode was established and a small Toxoplasma oocyst was recognized in cat feces (reviewed by Frenkel 1973). Toxoplasma is now classified in the protozoan phylum Apicomplexa, as a cystforming, heteroxenous coccidian in the family Sarcocystidae, subfamily Toxoplasmatina (Frenkel1977). Toxoplasma differs from the genera Besnoitia, Hammondia, Cystoisospora, Sarcocystis, and Frenkelia by the characteristics of the cysts and the transmission cycle (Frenkel 1973, 1977; Frenkel et al. 1979).

Frequency. Naturally occurring toxoplasmosis with illness and sometimes death has been observed in gondis, rabbits, guinea pigs, neotropical primates, and pigeons. Asymptomatic infection has been observed in guinea pigs, rats, chickens, and goats. Cats used to be given a free run in some animal colonies to catch wild and accidentally escaped mice. Disposition of the feces of these cats was not considered important, and it is likely that some of the feces were deposited in grain bins and bedding. With improved practices in handling animal feed and bedding and exclusion of pet and stray cats, the opportunity for infection can be diminished. Experimental toxoplasI?osis has been widely studied in mice, rabbits, and hamsters: these animals are more susceptible than rats and guinea pigs, which develop an age-dependent resistance. In these laboratory studies, direct transmission does not occur from animal to animal except by occasional cannibalism and congenital infection. However, experimental infection with oocysts from cat feces must be conducted with care, because a portion of the fed oocysts may be passed intact in the feces, where they will withstand drying and are thus capable of infecting other animals. Bedding should be changed and autoclaved 1 day and 2 days after the administration of oocysts. Food and bedding are the fomites, there is no evidence of airborne infection.


Generalized toxoplasmosis is similar in animals and man, keeping in mind their different degrees of resistance and strains of Toxoplasma which differ in pathogenicity. Well-adapted laboratory strains may convey a distorted impression, killing mice 3-4 days after intraperitoneal inoculation of a large number of organisms. However, of 31 recent natural isolates, 90% gave rise to inapparent infections in mice (Ruiz and Frenkel 1980) and fatal infections could be transformed to chronic infections when mice were treated prophylactically with 15-60 mg% of sulfadiazine or sulfamerazine in the drinking water. Adult rats are resistant even to strains pathogenic to mice, as are adult guinea pigs, adult humans, and most children. Ocular toxoplasmosis in man has been reproduced in hamsters; after subcutaneous inoculation, followed by sulfadiazine treatment, the retinas of many of these animals became infected (Frenkel 1961). Relapsing chronic toxoplasmosis seen in humans has also been reproduced in hamsters treated with pharmacologic doses of corticosteroids or cyclophosphamide (Frenkel et al. 1978). Most experimental infections are produced by injection, the intraperitoneal route being much more effective than the subcutaneous. Oral infection is the usual route in animals and man, either with oocysts from cat feces or with tissue cysts from infected meat (Dubey and Frenkel 1973). The course of the disease and the development of immunity may differ according to the route of infection.

References

Chobotar B, Scholtyseck E (1982) Ultrastructure. In: Long PL (ed) The biology of the coccidia. University Park Press, Baltimore, chap 4

Dubey JP, Frenkel JK (1973) Experimental toxoplasma infection in mice with strains producing oocysts. J Parasito159: 505-512

FrenkeIJK (1961) Pathogenesis of toxoplasmosis with a consideration of cyst rupture in Besnoitia infection. Surv Ophthalmol6: 799-825

Frenkel JK (1973) Toxoplasma in and around us. Bioscience 23: 343-352

FrenkeIJK (1977) Bensoitia wallacei of cats and rodents: with a reclassification of other cyst-forming isosporoid coccidia. J Parasitol 63: 611-628

Frenkel JK, Amare M, Larsen W (1978) Immune competence in a patient with Hodgkin's disease and relapsing toxoplasmosis. Infection 6 (2): 84-91

FrenkelJK, HeydornAO, Mehlhorn H, Rommel M (1979) Sarcocystinae: Nomina dubia and available names. Z Parasitenkd 58: 115-139

Ruiz A, FrenkeIJK (1980) Intermediate and transport hosts of Toxoplasma gondii in Costa Rica. Am J Trop Med Hyg 29: 1161-1166

Subject Index*

A, mice, 106t mouse strain, 148t

Acatalasemic, mouse, 150t Acetate, cortisone, 218, 220f Acetoxypropylnitrosamine, APPN, 28t ACI N, rat, 115t Acrylate, ethyl, 46t Adenocarcinoma, 47, 50f, 112

anterior nasal epithelium rat, 67 ethmoid region, 49f, 50f ethomoturbinal, 48f lung hamster, 132t

mouse metastasis renal, 145f maxilloturbinal, 67f nasoturbinal, 68f type B, lung mouse metastatic

mammary, 143f Adenoma, 41

alveologenic, 99,102 bronchiolo-alveolar, 102, 108 cell, type II, l04f Clara cell, 108f, 108, 110f lung hamster, 132t

mouse, alveolar type II cell, 102 bronchiolar, 108 type II, 102f, 103f, 104f

cell, 105f rat, bronchiolar alveolar, 99, 100f

maxilloturbinate rat, polypoid, 43f nasal cavity rat, 46t

mucosa rat, polypoid, 41 nonciliated light cell, 45f nose rat, polypoid, 42f papillary, 41, 108, 110f, Illf polypoid,41f pulmonary, 99,102 rat, polypoid nasal, 44f type II, 103f

cell,102 Adenomatosis, bronchioalveolar, 177

pulmonary, 177 Adenomatous polyp, 33, 41 Adenosquamos carcinoma lung

hamster, 132t Aeruginosa, Pseudomonas, 215 AlBrA mouse strain, 148t Alkylnitrosamines, 122 Alveolar cell carcinoma, 112

rat, type II, 96f epithelialization, 177 exudate bleomycin, lung mouse,

161f histiocytosis rat, 169

hyperplasia, 177 lipoproteinosis, lung rat, 173f, 174f

rat, 171 lung, 171f, 172f

macrophage, asbestos body, 184f proteinosis, 171 squamous metaplasia, 199f type II cell adenoma lung mouse,

102 Alveoli hamster lung, 96f Alveologenic adenoma, 99,102

carcinoma lung mouse, 146f Alveolus bleomycin, fibrosis lung

mouse, 163f giant cells hair particles, 191f hair particles pulmonary, 191f

Amelanotic melanoma mouse, pulmonary metastasis, 139f

Anaplastic carcinoma lung hamster, 120f

Anatomy nasal cavity rat, 3 Angiectasis,74t Anterior nasal cavity, squamous cell

carcinoma, 56f epithelium rat, adenocarcinoma,

67 Anthracosis, 180 APPN acetoxypropylnitrosamine, 28t APUD-type cells, neuroendocrine,

121f Arm, dynein, 92f Aromatic hydrocarbons, halogenated,

201 PAHs Polycyclic, 28t

Artery embolic hair, pulmonary, 189f lung mouse mammary tumor emboli

pulmonary, 141f thrombotic pulmonary, 187f

Asbestos bodies, 185f body, 186f

alveolar macrophage, 184f cholesterol clefts, 185f inhalation, 184f pneumoconiosis, 183

Asbestosis hamster, 183 Ash hamster, fly, 182t

pneumoconiosis hamster, fly, 180 Aspergillosis mucormycosis lung rat,

224 rat lung, 224f

Aspergillus terreus pleura rat, 225f Auratus, Mesocricetus, 33 Axon, intraepithelial, 8f

B6C3 Fl mice, 74t endogenous lipid pneumonia, 166

mouse, 148t, 149t BaA Benz(a)anthracene, 28t Baboon, pulmonary toxicity bleomycin, 165t

BALB/c C, mice, 106t C3H/He mouse strain, 148t cfC3H/Cbl Se mouse strain, 148t cfC3H mouse strain, 148t CfRIII mouse strain, 148t cNIV mouse strain, 148t cStCrl, mouse, 149t, 150t

BaP Benzo(a)pyrene, 28t N-Nitrosobis acetoxypropyl amine, 28t

Basal lamina, 8f Benz(a)anthracene, BaA, 28t Benzo(a)pyrene, 122

BaP,28t Benzo(e)pyrene, BeP, 28t BeP Benzo e pyrene, 28t Besnoitia, 230 BHP, 63f, 65f, 122

N-Nitrosobis hydroxypropyl amine, 28t

Biochemistry hypophysectomized rat, serum,169t

Bladder, metastasis lung urinary, 150t Bleeding, tail veins, 188t, 193t Bleomycin baboon, pulmonary toxicity, 165t

dog, pulmonary toxicity, 165t fibrosis lung mouse, 162f

alveolus, 163f hamster, pulmonary toxicity, 165t injury mouse pulmonary fibrosis, 160

lung mouse alveolar exudate, 161f pulmonary blood vessels, 162f vascular lesion, 161f

mouse, pulmonary toxicity, 165t pheasant, pulmonary toxicity, 165t

Blood vessels bleomycin, lung mouse pulmonary, 162f

Bodies, asbestos, 185f Body alveolar macrophage, asbeStos,

184f . asbestos, 186f giant cells, foreign, 189f granuloma foreign, 189f hamster lung, neuroepithelial, 93f

Bordetella bronchiseptica, 215

* Note: Page numbers in boldface indicate the principal discussion; figures are designated by the letter "P' following the page number; tables are found on page numbers followed by the letter "t"

232 Subject Index

Bronchial epithelium, serous cell rat, 90f

lymphoid tissue rat, normal, 215f Bronchiectasis, enzootic, 213 Bronchioalveolar adenomatosis, 177 Bronchiolar adenoma lung mouse, 108

alveolar adenoma lung rat, 99, 100f carcinoma lung rat, 112, 114f

rat,l13f hyperplasia F344 rat, 179t

lung rat, 177f, 177, 178f tumors rats, naturally occuring,

115t carcinoma, 112 epithelium, proliferation, 185f metaplasia, 177

Bronchiole multiple epithelial syncytia, 196f

murine respiratory mycoplasmosis, 216f

rat lung brush cell, 94f Clara cell, 94f

Bronchiolization lung hamster, 181f Bronchiolo-alveolar adenoma, 102, 108

Bronchiseptica, Bordetella, 215 Bronchus hamster, 121f

murine respiratory mycoplasmosis, 216f

Brush cell, 7f, 9f bronchiole rat lung, 94f

Bulla purulent exudate, tympanic, 80f

C3H/Cb/Se mouse strain, 148t He mouse strain, BALB/C, 148t mice,106t mouse strain, 148t

C3HeB/FEJ hybrid, mouse, 149t C57BLl6J mouse strain, 148t C57 black, mice, 106t

leaden L or M, mice, 106t C57BL/6J mouse, squamous cell carcinoma pulmonary vein, 130f

mouse strain, 148t C57BLl6Jx, mouse, 149t Candida turolopsis glabrata, 219, 222f Carcinogens respiratory epithelium

syrian golden hamster, 33 Carcinoma, 112

alveolar cell, 112 anterior nasal cavity, squamous cell,

56f bronchiolar, 112, epidermoid, 54, 62, 117 in situ, dysplasia, 34 larynx, clear cell, 75f, 76f

syrian hamster, clear cell, 75 lung F344 rat, squamous cell, 129f

hamster, adenosquamous, 132t anaplastic, 120f squamous cell, 119f, 132t

mouse, alveologenic, 146f hepatocellular, 148t metastasis harderian gland,

145f

renal transitional cell, 145f metastatic hepatocellular, 143f

rat, bronchiolar alveolar, 112, 114f epidermoid, 124, 127 naturally occurring squamous

cell,126t radiation-induced squamous

cell, 127 squamous cell, 124p, 125f, 128f

syrian hamster, squamous cell, 117 mouse mammary gland, 148t nasal,60f

cavity rat, squamous cell, 58f mucosa rat, squamous cell, 54, 59t

squamous cell, 55f nasoturbinate rat, squamous cell, 57f pulmonary vein C57Bl J mouse, squamous cell, 130f

rat, bronchiolar alveolar, 113f lung, squamous cell, 124 mouse hamster man, comparison

squamous cell, 132t squamous cell, 47, 63f, 64f, 65f upper respiratory tract syrian

hamster, squamous ce, 62 Carinii, Pneumocystis, 221f, 223

rat lung Pneumocystis, 220f Catarrh, infections, 78 Caudal vein male rat, thrombus, 190f CBA mouse strain, 148t Cell adenoma, Clara, 108f, 108, 110f

lung mouse, alveolar type II, 102 type II, 105f

nonciliated light, 45f type II, 102

bronchiole rat lung brush, 94f Clara,94f

brush, 7f, 9f carcinoma, alveolar, 112

anterior nasal cavity, squamous, 56f

larynx, clear, 75f, 76f syrian hamster, clear, 75

lung F344 rat, squamous, 129f hamster squamous, 119f, 132t mouse metastasis renal transitional, 145f

rat, naturally occurring squamous, 126t

radiation-induced squamous, 127

squamous, 124, 125f, 128f syrian hamster, squamous, 117

nasal cavity rat, squamous, 58f mucosa rat, squamous, 54p, 59t

squamous, 55f nasoturbinate rat, squamous, 57f pulmonary vein C57Bl J mouse,

squamous, 130f rat lung, squamous, 124

mouse hamster man, comparison squamous, 132t

squamous, 47, 63f, 64f, 65f upper respiratory tract syrian

hamster, squamo, 62

ciliated, 9f, 20f Clara,111f granuloma hair particle, lung giant,

193t lung giant, 188t, 193t skin particle, lung giant, 193t

hyperplasia, goblet, 30 mucous,30f

lung mouse type II epithelial, 163f Mycoplasma pulmonis, tracheal epithelial, 82f

nonciliated columnar, 7f, 9f dark,44f

papilloma, squamous, 33 pneumonia, foamy, 166

interstitial plasma, 218 rat bronchial epithelium, serous, 90f

type II alveolar, 96f secretory, 26f tumor, lung mouse metastasis

ovarian granulosa, 144f squamous,62,117 type I, 14f, 24f, 25f

II, 14f, 19f adenoma, 104f ciliated, 16f, 18f

Cells, cuboidal, 7f differentiation ciliated, 17

mucous, 26 mucous, 20

epithelial, 12f foreign body giant, 189f hair particles alveolus, giant, 191f hair-phagocytosing giant, 192t malignant squamous, 64f, 65f, 118f neuroendocrine APUD-type, 121f type I mucous, 22f

CF-l, mouse, 149t, 150t strain, 148t

Chemically induced lung tumors F344 rats,101t

Chloride, dimethy1carbamoyl, 59t hydrogen, 59t

Chlormethyl ether, 59t Cholesterol clefts asbestos, 185f

granulomas, lung rat, 174f pneumonia, 166

CHR chrysene, 28t Chronic murine pneumonia, 213

respiratory mycoplasmosis, 81f respiratory disease, murine, 78, 213

Crysene, CHR, 28t Ciliated cell, 9f, 20f

type II, 16f, 18f cells, differentiation, 17 mucous cells, differentiation, 26

Cilium, human respiratory, 92f Clara cell, ll1f

adenoma, 108, 108f, 110f bronchiole rat lung, 94f

Clear cell carcinoma larynx, 75f, 76f syrian hamster, 75

Clefts asbestos, cholesterol, 185f Coliforms,221 Columnar cell, nonciliated, 7f, 9f

Comparison squamous cell carcinoma rat mouse hamster man, 132t

Coronaviridae, 86, 212 Coronavirus infection, 86

lung, rat, 204f rat, 203

Parker's rat, 203 interstitial pneumonia rat, 204f

Coronaviruses, mouse, 201 Corticosteroids, 219, 230 Cortisone acetate, 218, 220f Corynebacterium kutscheri, 215, 221

mouse, 201 rat, 201

Cruzi, Trypanosoma, 227 Cs, mouse, 150t Cuboidal cells, 7f Cyclophosphamide, 219, 230 Cyst, pneumocystis, 222f Cystoisopora, 230

Dark cell, nonciliated, 44f Dba, mice, 106t

mouse strain, 148t xC57BL Fl, mouse, 149t

DBF1, mouse, 149t DBN,122

N-Nitrosodibuthylamine, 28t N-nitrosodiethylamine diethylnitrosamine, 28t

Desquamative interstitial pneumonia, 171

pneumonia, 171 DHPN Dihydroxy-di-n-propylnitrosamine, 28t

Diagnosis papillary tumors, differential, 39

Dibromo-3-chloropropane,51f Dibromochloropropane, 67f Dibromoethane,101t Diethylnitrosamine, DEN

N-nitrosodiethylamine,28t Differential diagnosis papillary tumors, 39

Differentiation, 13 ciliated cells, 17

mucous cells, 26 mucous cells, 20

Dihydroxy-di-n-propylnitrosamine, DHPN,28t

Dimethyl sulfoxide, DMSO, 28t Dimethyl-l benzanthracene, 122

DMBA,28t Dimethylcarbamoyl chloride, 59t Dinitrosopiperazine, 46t Dioxane, 59t Disease, murine chronic respiratory, 78,213

DMBA 9,1 O-Dimethyl-l ,2-benzanthracene, 28t

DMDPN N-Nitrosobis (2-methylpropyl) amine, 28t

DMSO Dimethyl sulfoxide, 28t Dog, pulmonary toxicity bleomycin, 165t

DPN,64f N-Nitrosodi-n-propylamine,28t

Duct, nasolacrimal, 4f Dynein arm, 92f Dysplasia carcinoma in situ, 34

trachea, 35f Dysplastic epithelium, 29f

squamous epithelium, 63f

Emboli lung, skin, 186 pulmonary artery, lung mouse

mammary tumor, 141f vascular system, hair fragment,

186 Embolic hair, 187f

fragment lung, 193f pulmonary artery, 189f

Embolism hairs, pulmonary, 186 pulmonary hair, 186

Encephalitozoon, 227 Endogenous lipid pneomonia, 171

B6C3Fl mice, 166 mouse, 166f, 167f

Enzootic bronchiectasis, 213 Epichlorohydrin, 59t Epidermoid carcinoma, 54, 62, 117

lung rat, 124, 127 metaplasia, 30 papillary tumor, 33

Epithelia, olfactory, 6f respiratory, 6f

Epithelial cell, lung mouse type II, 163f Mycoplasma pulmonis, tracheal, 82f

cells,12f papilloma, 33 syncytia, bronchiole multiple, 196f

Epithelialization, alveolar, 177 Epithelioid mesothelioma hamster, 134f, 135f, 136f

Epithelium, dysplastic, 29f squamous, 63f

hamster, tracheal, l1f olfactory, 9f proliferation bronchiolar, 185f rat, adenocarcinoma anterior nasal, 67

trachea, 89f respiratory, 6f, 7f, 8f, 9f, 36 serous cell rat bronchial, 90f syrian golden hamster, carcinogens

respiratory, 33 tracheal, 11

tracheal, 13f, 15f, 21f, 24f Esthesioneuroblastoma, 47 Ether, chlormethyl, 59t

phenylglycidyl, 59t Ethmoid region, adenocarcinoma, 49f,

50f turbinate, 4f turbinates rat, neoplasms mucosa,

47 Ethomoturbinal, adenocarcinoma, 48f Ethyl acrylate, 46t Explant culture fetal trachea, 28

Subject Index 233

cultures fetal trachea syrian golden hamsters, 27

fetal trachea, 30f tracheal, 29f

Exudate bleomycin, lung mouse alveolar,161f tympanic bulla purulent, 80f

F344, rat, 115t, 126t bronchiolar alveolar hyperplasia,

179t olfactory region, 52f, 53f squamous cell carcinoma lung,

129f rats, chemically induced lung tumors,10H

FC3H/Nctr, mouse, 149t, 150t Fetal trachea, 30f

explant, 30f culture, 28

syrian golden hamsters, explant cultures, 27

tracheal explant, 29f Fibrosarcoma lung mouse, 13H

rat, 131t, 131t Fibrosis, bleomycin injury mouse

pulmonary, 160 lung mouse alveolus bleomycin,

163f bleomycin, 162f

Fly ash hamster, 182t pneumoconiosis hamster, 180

Foamy cell pneumonia, 166 Foreign body giant cells, 189f

granuloma, 189f Formaldehyde, 46t, 57f, 59t, 59t

nasoturbinate rat, 43f Frenkelia, 230 Frequency pulmonary tumors mice, 106t

Function lung, structure, 89 Fungal hyphae, lung, 225f

yeasts, 222f Fungi,221 Fusocellular mesothelioma hamster, 135f

Gases, oxidant, 201 Giant cell granuloma hair particle, lung, 193t

lung, 188t, 193t skin particle, lung, 193t

cells, foreign body, 189f hair particles alveolus, 191f hair-phagocytosing, 192t

Giardia, mouse, 201 Glabrata, Candida torulopsis, 219,

222f Gland carcinoma, lung mouse

metastasis harderian, 145f mouse mammary, 148t

metastasis lung harderian, 150t mammary,15Ot

virus infection, rat submaxillary, 84, 210

234 Subject Index

Glands rat trachea, seromucous, 91f Goblet cell hyperplasia, 30 Golden hamster, carcinogens respira

tory epithelium syrian, 33 tracheal epithelium, syrian, 11

hamsters, explant cultures fetal trachea syrian, 27

Gondii, Toxoplasma, 227, 229 GR mouse strain, 148t Granuloma foreign body, 189f

hair particle, lung giant cell, 193t lung giant cell, 188t, 193t skin particle, lung giant cell, 193t

Granulomas, lung rat cholesterol, 174f Granulosa cell tumor, lung mouse

metastasis ovarian, 144f

Hair, embolic, 187f embolism, pulmonary, 186 fragment emboli pulmonary

vascular system, 186 lung, embolic, 193f

intravenous injection, 191f particle, lung giant cell granuloma,

193t thromboarteritis, 193t

particles alveolus, giant cells, 191 f lung, 188t pulmonary alveolus, 191f

pulmonary artery embolic, 189f segment, pigmented, 187f tail veins thrombophlebitis, 193t thromboarteritis, tail veins, 188t vessel, tail veins, 193t

Hair-phagocytosing giant cells, 192t Hairs, pulmonary embolism, 186 Halogenated aromatic hydrocarbons,

201 Hammondia, 230 Hamster, adenocarcinoma lung, 132t

adenoma lung, 132t adenosquamous carcinoma lung,

132t Anaplastic carcinoma lung, 120f asbestosis, 183 bronchiolization lung, 181f bronchus, 121f carcinogens respiratory epithelium

syrian golden, 33 clear cell carcinoma larynx syrian, 75

epithelioid mesothelioma, 134f, 135f,136f

fly ash, 182t pneumoconiosis, 180

fusocellular mesothelioma, 135f hyperplasia lung, 181f lung, alveoli, 96f

interalveolar septum, 96f neuroepithelial body, 93f

man, comparison squamous cell carcinoma rat mouse, 132t

papillary mesothelioma, 134f mucoepidermoid tumor trachea, 33f

peripheral lung, 95f pleural mesothelioma syrian, 133 pneumoconiosis lung, 180f pulmonary toxicity bleomycin, 165t radioactive materials, lung tumor

syrian, 132t squamous cell carcinoma, lung,

119f,132t syrian, 117

upper respiratory tract syri, 62

metaplasia, lung, l18f toxoplasmic pneumonia, 228f toxoplasmosis lung mouse, 227 trachea, 22f tracheal epithelium, 11 f

syrian golden, 11 Hamsters, explant cultures fetal trachea syrian golden, 27

Harderian gland carcinoma, lung mouse metastasis, 145f

metastasis lung, 150t Hemagglutinating virus infection JHV, Japanese, 195

mouse HVM, 195 Japan HVJ, 195

Hemangioma,74t Hemangiosarcoma,74t

lung mouse, 131t rat,131t

nasal cavity, 73f, 74f mouse, 72

Hepatoblastoma, lung mouse, 148t Hepatocellular carcinoma, lung mouse, 148t

metastatic, 143f Hepatoma, lung mouse, 148t HEPES 4 (2-Hydroxyethyl)-1-piperazineethane sulfonic acid, 28t

Hexamethylphosphoramide, 46t, 59t Histiocytosis, multifocal, 171

rat, alveolar, 169 Histoplasma, 227 Holtzman-SD, rat, 115t HPPN N-Nitroso-2-hydroxypropyl-n

propylamine, 28t Human respiratory cilium, 92f HVJ, hemagglutinating virus Japan,

195 HVM, hemagglutinating virus infection mouse, 195

Hybrid, mouse C3HeB FEJ, 149t Hydrocarbons, halogenated aromatic, 201

PAHs Polycyclic aromatic, 28t Hydrogen chloride, 59t Hydroxyethyl piperazineethane sulfonic acid, HEPES, 28t

Hyperplasia, 34 alveolar, 177 F344 rat, bronchiolar alveolar, 179t goblet cell, 30 lung hamster, 181f

rat, bronchiolar alveolar, 177f, 177, 178f

mucous cell, 30f trachea,35f

Hyphae, lung fungal, 225f Hyphomycosis, 224 Hypophysectomized rat, serum biochemistry, 169t

I, mice, 106t Induced tumors mice, pulmonary

metastasis, 149t Infection, coronavirus, 86

JHV, Japanese hemagglutinating virus, 195

lesions due to, 195 lung mouse rat, pneumonia virus

mice, 206 sendai virus, 195

sialodacryoadenitis virus, 210 rat coronavirus, 204f

rat coronavirus, 203 mouse HVM, hemagglutinating virus, 195

parainfluenza virus, 195 Parkers rat coronavirus, 203 rat submaxillary gland virus, 84, 210 reparative phase, sendai virus, 197f resolution phase, sendai virus, 200f SDAV, 84, 210 sendai virus, 196f, 197f upper respiratory tract rat, sialo

dacryoadenitis vi, 84 Infections, 78

catarrh, 78 Inhalation asbestos, 184f Injection hair, intravenous, 191f Injury mouse pulmonary fibrosis, bleomycin, 160

Interalveolar septum hamster lung, 96f Interstitial plasma cell pneumonia, 218

pneumonia, desquamative, 171 PVM rat, 208f rat coronavirus, 204f

Intraepithelial axon, 8f Intravenous injection hair, 1911'

Japan HVJ, hemagglutinating virus, 195 Japanese hemagglutinating virus

infection JHV, 195 JHV, Japanese hemagglutinating virus

infection, 195 Jiroveci, Pneumocystis, 223

K virus, mouse, 201 Klebsielle pneumoniae, 215 Kutscheri, Corynebacterium,/215, 221

mouse Corynebacterium, 201 rat Corynebacterium, 201

Lamina, basal, 8f Larynx, clear cell carcinoma, 75f, 76f

syrian hamster, clear cell carcinoma, 75

Leishmania spp, 227 Lesion bleomycin, lung mouse vascular, 161f

Lesions due to infection, 195 lung, nonneoplastic, 160 microembolic pulmonary, 186 tail veins lung, 188t

Sprague-Dawley rat, 193t Light cell adenoma, nonciliated, 45f Lipid pneumonia B6C3Fl mice, endogenous, 166

endogenous, 171 mouse, endogenous, 166f, 167f

Lipidosis rat, pulmonary, 169t, 169, 170f

Lipoproteinosis, lung rat alveolar, 173f, 174f

rat, alveolar, 171 lung alveolar, 171f, 172f

Liver, metastasis lung, 149t Lung, 87

alveolar lipoproteinosis rat, 171f, 172f

alveoli hamster, 96f aspergillosis, rat, 224f brush cell, bronchiole rat, 94f Clara cell, bronchiole rat, 94f embolic hair fragment, 193f F344 rat, squamous cell carcinoma, 129f

fungal hyphae, 225f giant cell granuloma, 188t, 193t

hair particle, 193t skin particle, 193t

hair particles, 188t hamster, adenocarcinoma, 132t

adenoma, 132t adenosquamos carcinoma, 132t anaplastic carcinoma, 120f bronchiolization,181f hyperplasia, 181f peripheral,95f pneumoconiosis, 180f squamous cell carcinoma, 119f,

132t metaplasia, 118f

harderian gland, metastasis, 150t interalveolar septum hamster, 96f lesions tail veins, 188t liver, metastasis, 149t mammary gland, metastasis, 150t metastasis, 138 mouse alveolar exudate bleomycin,

161f type II cell adenoma, 102

alveologenic carcinoma, 146f alveolus bleomycin, fibrosis, 163f bleomycin, fibrosis, 162f bronchiolar adenoma, 108 fibrosarcoma, 131 t hamster, toxoplasmosis, 227 hemangiosarcoma, 1311 hepatoblastoma, 148t hepatocellular carcinoma, 148t hepatoma, 148t lymphosarcoma,131t mammary tumor emboli pulmonary artery, 141f

metastasis harderian gland carcinoma, 145f

malignant schwannoma, 146f ovarian granulosa cell tumor,

144f renal adenocarcinoma, 145f

transitional cell carcinoma, 145f

subcutaneous sarcoma, 141f metastatic hepatocellular

carcinoma, 143f mammary adenocarcinoma type B, 143f

tumors, 138 pleural metastasis osteosarcoma,

146f pulmonary blood vessels bleomycin, 162f

rat, pneumonia virus mice infection, 206

sendai virus infection, 195 sialodacryoadenitis virus

infection, 210 thrombi malignant schwannoma,

146f type II adenoma, 102f, 103f, 104f

cell adenoma, 105f epithelial cell, 163f

vascular lesion bleomycin, 161f neuroepithelial body hamster, 93f nonneoplastic lesions, 160 ovary, metastasis, 150t Pneumocystis carinii, rat, 220f pneumonia virus mice rat, 207f rat alveolar lipoproteinosis, 173f,

174f aspergillosis mucormycosis, 224 bronchiolar alveolar adenoma, 99,

100f carcinoma, 112, 114f hyperplasia, 177f, 177, 178f

cholesterol granulomas, 174f coronavirus infection, 204f epidermoid carcinoma, 124, 127 fibrosarcoma, 131t, 131t hemangiosarcoma,131t lymphosarcoma,131t mesothelioma, 131 t mucormycosis, 226f murine respiratory

mycoplasmosis, 213 mycoplasmosis,215f naturally occurring squamous cell

carcinoma, 126t normal, 214f pneumocystosis, 218f, 218 radiation-induced squamous cell

carcinoma, 127 rat coronavirus infection, 203 reticulosarcoma,131t squamous cell carcinoma, 124,

125f,128f RIll mouse metastatic mammary tumor, 140f

secondary tumors, 138

Subject Index 235

skin emboli, 186 metastasis, 150t particles, 188t

spleen, metastasis, 150t Sprague-Dawley rat, lesions tail veins, 193t

squamous cell carcinoma rat, 124 structure function, 89 syrian hamster, squamous cell

carcinoma, 117 thromboarteritis, 188t, 193t

hair particle, 193t skin particle, 193t

tumor mice radioactive materials, 131t

rat, 1311 syrian hamster radioactive

materials, 132t tumors F344 rats, chemically

induced, 1011 urinary bladder, metastasis, 150t

Lymphoid tissue rat, normal bronchial, 215f

Lymphosarcoma lung mouse, 131t rat,131t

M-2-0B N-Nitrosomethyl (2-oxobutyl) amine, 28t

Macrophage, asbestos body alveolar, 184f

Male rat, thrombus caudal vein, 190f Malignant mesothelioma, 133

schwannoma, lung mouse metastasis, 146f

thrombi,146f squamous cells, 64f, 65f, 118f

Mammary adenocarcinoma type B, lung mouse metastatic, 143f

gland carcinoma, mouse, 148t metastasis lung, 150t

tumor,138f emboli pulmonary artery, lung

mouse,141f lung, RIll mouse metastatic, 140f

Man, comparison squamous cell carcinoma rat mouse hamster, 132t

Mass, rat subcutaneous, 55f Materials, lung tumor mice radioactive, 1311

syrian hamster radioactive, 132t radioactive,131t

Maxilloturbinal, adenocarcinoma, 67f Maxilloturbinate, 4f

rat, polypoid adenoma, 43f Melanoma mouse, pulmonary metastasis amelanotic, 139f

pulmonary metastasis melanotic, 139f

Melanttic melanoma mouse, pulmonary metastasis, 139f

Mesocricetus auratus, 33 Mesothelial neoplasia, 133 Mesothelioma hamster, epithelioid,

134f, 135f, 136f fusocellular, 135f

236 Subject Index

Mesothelioma hamster papillary, 134f

lung rat, 131t malignant, 133 syrian hamster, pleural, 133

Metaplasia, alveolar squamous, 199f bronchiolar, 177 epidermoid, 30 lung hamster squamous, 118f simple, 35 squamous, 29f, 30f, 34, 36

Metastasis amelanotic melanoma mouse, pulmonary, 139f harderian gland carcinoma, lung

mouse, 145f induced tumors mice, pulmonary,

149t lung, 138

harderian gland, 150t liver, 149t mammary gland, 150t ovary,150t skin, 150t spleen, 150t urinary bladder, 150t

malignant schwannoma, lung mouse, 146f

melanotic melanoma mouse, pulmonary, 139f

osteosarcoma, lung mouse pleural, 146f

ovarian granulosa cell tumor, lung mouse,l44f

renal adenocarcinoma, lung mouse, 145f transitional cell carcinoma, lung

mouse, 145f subcutaneous sarcoma, lung mouse,

141f untreated mice, pulmonary, 148t

Metastatic hepatocellular carcinoma, lung mouse, 143f mammary adenocarcinoma type B, lung mouse, 143f tumor lung, RIll mouse, 140f

tumors lung mouse, 138 Methylnitrosourea, MNU, 28t Mice A, 106t

B6C3Fl,74t BALBI c C, 106t C3H,106t C57 black, 106t

leaden, Lor M, 106t dba,106t endogenous lipid pneumonia

B6C3F1,166 frequency pulmonary tumors, 106t 1,106t infection lung mouse rat,

pneumonia virus, 206 pneumonia virus, 209 pulmonary metastasis induced tumors, 149t

untreated 148t PVM, pneumonia virus, 206

radioactive materials, lung tumor, 131t

rat lung, pneumonia virus, 207f Swiss,106t

Microembolic pulmonary lesions, 186 Mixed polyp, 33 MNU Methylnitrosourea, 28t Moniliformis, Streptobacillus, 215, 221 MOP N-Nitrosomethyl oxopropyl amine,28t

Mouse acatalasemic, 150t alveolar exudate bleomycin, lung,

161f type II cell adenoma lung, 102

alveologenic carcinoma lung, 146f alveolus bleomycin, fibrosis lung,

163f B6C3F1,149t BALB/cStCr1, 149t, 150t bleomycin, fibrosis lung, 162f bronchiolar adenoma lung, 108 C3HeB/FEJ hybrid, 149t C57BLl6Jx,149t CF-l, 149t, 150t coronariruses, 201 Corynebacterium kutscheri, 201 Csb, 150t DBAxC57BLlF1,149t DBF1,149t endogenous lipid pneumonia, 166f,

167f fC3H Nctr, 149t, 150t fibrosarcoma lung, 131t giardia, 201 hamster man, comparison squa

mous cell carcinoma rat, 132t toxoplasmosis lung, 227

hemangiosarcoma lung, 131t nasal cavity, 72

hepatoblastoma, lung, 148t hepatocellular carcinoma, lung, 148t hepatoma, lung, 148t HVM, hemagglutinating virus infection, 195

K virus, 201 lymphosarcoma lung, 131t mammary gland carcinoma, 148t

tumor emboli pulmonary artery, lung, 141f

metastasis harderian gland carcinoma, lung, 145f

malignant schwannoma, lung, 146f

ovarian granulosa cell tumor, lung,l44f

renal adenocarcinoma, lung, 145f transitional cell carcinoma,

lung, 145f subcutaneous sarcoma, lung, 141f

metastatic hepatocellular carcinoma, lung, 143f mammary adenocarcinoma type

B, lung, 143f tumor lung, RIll, 140f

tumors lung, 138

Mycoplasma pulmonis, 201 pleural metastasis osteosarcoma,

lung, 146f pneumocystis, 201 pneumonia virus, 201, 206 pulmonary metastasis amelanotic melanoma, 139f

pulmonary blood vessels bleomycin, lung, 162f fibrosis, bleomycin injury, 160 metastasis melanotic melanoma,

139f toxicity bleomycin, 165t

rat, pneumonia virus mice infection lung, 206

sendai virus infection lung, 195 sialodacryoadenitis virus infection

lung, 210 spironucleus, 201 squamous cell carcinoma pulmo

nary vein C57Bl J, 130f strain, A, 148t, 148t

AlBrA,148t B6C3HF1, 148t BALB/C/C3H/He,148t

cfC3H/Cb/Se,148t cfC3H,148t cfRIII,148t cNIV,148t

C3H/HE,148t Cb/Se,148t

C57BLl6J,148t CBA,148t Cf-l,148t DBA, 148t OR, 148t NIH white, 148t RIll, 148t

Dm/Se,148t thrombi malignant schwannoma,

lung, 146f toxoplasma, 201 toxoplasmic pneumonia nude, 228f type II adenoma lung, 102f, 103f,

104f cell adenoma lung, 105f epithelial cell, lung, 163f

vascular lesion bleomycin, lung, 161f

MPN N- Nitrosomethyl-n-propylamine,28t

Muco-epidermoid papillary tumor, 36f Mucoepidermoid papillary tumor, 33

respiratory tumor, 39f tumor trachea hamster, pal?illary, 33f

Mucormycosis lung rat, 226£ aspergillosis, 224

Mucosa ethmoid turbinates rat, neoplasms, 47

normal nasal septal, 79f rat, polypoid adenoma nasal, 41

squamous cell carcinoma nasal, 54,59t

tracheal, 81f Mucous cell hyperplasia, 30f

cells, differentiation, 20 ciliated, 26

type J, 22f Multifocal histiocytosis, 171 Multiple epithelial syncytia, bronchiole,196f

Murine chronic respiratory, disease, 78,213

pneumonia, chronic, 213 respiratory mycoplasmosis, 79f

bronchiole, 216f bronchus,216f chronic,81f lung rat, 213 rat, 213f, 214f

mycoplasmosis upper respiratory tract rat, 78

Mycoplasma pulmonalis, 215 pulmonis, 78, 82, 217

mouse, 201 rat, 201 tracheal epithelial cell, 82f

Mycoplasmosis, bronchiole murine respiratory,216f bronchus murine respiratory, 216f chronic murine respiratory, 81f lung rat, 215f

murine respiratory, 213 murine respiratory, 79f rat, murine respiratory, 213f, 214f

Mycoplasmosis upper respiratory tract rat, murine respiratory, 78

N-6-MI N-Nitrosohexamethyleimine,28t

N-nitroso-2-hydroxypropyl-n-propylamine, 122

HPPN,28t N -Nitrose-2-oxopropyl-n-propylamine, OPPN, 28t

N-nitrosobis (2-acetoxypropyl) amine, 122

N-Nitrosobis acetoxypropyl amine, BAP,28t (2-hydroxypropyl) amine, 122

BHP,28t (2-methylpropyl) amine, DMDPN,

28t N-nitrosodi-n-propylamine, 122

DPN,28t N-Nitrosodibuthylamine, DBN, 28t N-nitrosodiethylamine, 121f

diethylnitrosamine, DEN, 28t N-nitrosohexamethyleneimine,122

N-6-MI,28t N-Nitrosomethyl (2-oxobutyl) amine,

M-2-0B,28t (2-oxopropyl)amine,122

MOP,28t N-nitrosomethyl-n-propylamine, 122

MPN,28t N-nitrosomethylpiperazine, 49f, 52f N-Nitrosomorpholine, NM, 28t N-nitrosovinylethylamine, 122

YEN,28t

Nasal adenoma rat, polypoid, 44f carcinoma, 60f cavity, hemangiosarcoma, 73f, 74f

mouse, hemangiosarcoma, 72 rat,3f

adenoma, 46t anatomy, 3 squamous cell carcimona, 58f

squamous cell carcinoma anterior, 56f

epithelium rat, adenocarcinoma anterior, 67

mucosa rat, polypoid adenoma, 41 squamous cell carcinoma, 54,

59t squamous cell carcinoma, 55f

septal mucosa, normal, 79f Nasolacrimal duct, 4f Nasopharynx,4f Nasoturbinal, adenocarcinoma, 68f Nasoturbinate,4f

rat formaldehyde, 43f squamous cell carcinoma, 57f

Naturally occurring bronchiolar alveolar tumors rats, 115t

squamous cell carcinoma lung rat, 126t

Neoplasia, mesothelial, 133 Neoplasms, 33

mucosa ethmoid turbinates rat, 47 Neuroblastoma, olfactory, 47 Neuroendocrine APUD-type cells, 121f

Neuroepithelial body hamster lung, 93f

NIH white mouse strain, 148t Nitroacenaphthene, 101 t Nitrosamines, 63 Nitrosaminobutanone, 46t Nitrosomethylurea, NMU, 28t NM N-Nitrosomorpholine, 28t NMU Nitrosomethylurea, 28t Nonciliated columnar cell, 7f, 9f

dark cell, 44f light cell adenoma, 45f

Nonneoplastic lesions lung, 160 Normal bronchial lymphoid tissue rat, 215f

lung rat, 214f nasal septal mucosa, 79f rat trachea, 81f

Nose rat, polypoid adenoma, 42f Nude mouse, toxoplasmic pneumonia, 228f

Olfactory epithelia, 6f epithelium, 9f neuroblastoma, 47 organ, septal, 4f region F344 rat, 52f, 53f

tumor,51f OPPN,65f

N -Nitroso-2-oxopropyl-n-propylam,28t

Oregon, rat, 115t

Subject Index 237

Organ, septal olfactory, 4f vomeronasal, 4f

Osborne-Mendel, rat, 115t, 126t Osteosarcoma, lung mouse pleural

metastasis, 146f Ovarian granulosa cell tumor, lung

mouse metastasis, 144f Ovary, metastasis lung, 150t Oxidant gases, 201 Oxide, propylene, 74t

P-cresidine, 46t, 50f, 59t PAHs Polycyclic aromatic hydrocarbons, 28t

Papillary adenoma, 41, 108, 11 Of, ll1f mesothelioma hamster, 134f mucoepidermoid tumor trachea

hamster, 33f polyp, 33 tumor, epidermoid, 33

muco-epidermoid, 36f mucoepidermoid, 33 trachea,37f

tumors, 34 differential diagnosis, 39 ultrastructure, 37

Papilloma, 33, 41 epithelial, 33 squamous cell, 33

Parainfluenza virus, 202 infection, 195

Paramyxovirus, 202 Parker's rat coronavirus infection, 203 Particles alveolus, giant cells hair, 191f

lung hair, 188t skin, 188t

pulmonary alveolus, hair, 191f Pasteurella pneumotropica, 215, 221 Peripheral lung hamster, 95f Periphlebitis, tail veins, 188t, 193t Pheasant, pulmonary toxicity bleomycin, 165t

Phenacetin, 46t, 59t Phenylglycidyl ether, 59t Phlebitis, tail veins, 188t Phycomycosis, 224 Pigmented hair segment, 187f Plasma cell pneumonia, interstitial, 218 Pleura rat, Aspergillus terreus, 225f Pleural mesothelioma syrian hamster, 133

metastasis osteosarcoma, lung mouse, 146f

Pneumoconiosis, 180 asbestos, 183 hamster, fly ash, 180 lung hamster, 180f

Pneumocystis, 219, 227 carinii, 221f, 223

rat lung, 220f cyst, 222f jiroveci, 223 mouse, 201 trophozoites, 222f

Pneumocystosis lung rat, 218f, 218

238 Subject Index

Pneumonia B6C3Fl mice, endogenous lipid, 166

cholesterol, 166 chronic murine, 213 desquamative, 171

interstitial, 171 endogenous lipid, 171 foamy cell, 166 hamster, toxoplasmic, 228f interstitial plasma cell, 218 nouse, endogenous lipid, 166f, 167f nude mouse, toxoplasmic, 228f PVM rat, interstitial, 208f rat coronavirus, interstitial, 204f sialodacryoadenitis viral, 211 f virus, 206

mice, 209 infection lung mouse rat, 206 PVM,206 rat lung, 207f

mouse, 201, 206 Pneumoniae, Klebsielle, 215

rat Streptococcus, 201 Streptococcus, 215

Pneumotropica, Pasteurella, 215, 221

Pneumovirus, 209 Polycyclic aromatic hydrocarbons,

PAHs,28t Polyp, 33

adenomatous, 33, 41 mixed, 33 papillary, 33

Polypoid adenoma, 41f maxilloturbinate rat, 43f nasal mucosa rat, 41 nose rat, 42f

nasal adenoma rat, 44f tumor, 33

Predifferentiation stage, 11 Proliferation bronchiolar epithelium,

185f Propylene oxide, 74t Proteinosis, alveolar, 171 Proteus, 221 Pseudomonas, 221

aeruginosa,215 Pulmonary metastasis amelanotic melanoma mouse, 139f

Pulmonalis, Mycoplasma, 215 Pulmonary adenoma, 99,102

adenomatosis, 177 alveolus, hair particles, 191f artery embolic hair, 189f

lung mouse mammary tumor emboli, 141f

thrombotic, 187f blood vessels bleomycin, lung

mouse, 162f embolism hairs, 186 fibrosis, bleomycin injury mouse, 160

hair embolism, 186 lesions, microembolic, 186 lipidosis rat, 196t, 169, 170f

metastasis induced tumors mice, 149t

melanotic melanoma mouse, 139f untreated mice, 148t

toxicity bleomycin baboon, 165t dog, 165t hamster, 165t mouse, 165t pheasant, 165t

tumors mice, frequency, 106t vascular system, hair fragment

emboli, 186 vein C57BL/6J mouse, squamous

cell carcinoma, 130f Pulmonis, mouse Mycoplasma, 201

Mycoplasma, 78, 82, 217 rat Mycoplasma, 201 tracheal epithelial cell Mycoplas

ma,82f Purulent exudate, tympanic bulla, 80f PVM, pneumonia virus mice, 206

rat, interstitial pneumonia, 208f

Radiation-induced squamous cell carcinoma lung rat, 127

Radioactive materials, 131t lung tumor mice, 131t

syrian hamster, 132t Rat ACIIN, 115t

adenocarcinoma anterior nasal epithelium, 67

adenoma nasal cavity, 46t alveolar histiocytosis, 169

lipoproteinosis,171 lung, 173f, 174f

anatomy nasal cavity, 3 aspergillosis mucormycosis lung,

224 Aspergillus terreus pleura, 225f bronchial epithelium, serous cell, 90f bronchiolar alveolar adenoma lung, 99,100f

carcinoma,113f lung, 112, 114f

hyperplasia F344, 179t lung, 177, 177~ 178f

cholesterol granulomas, lung, 174f coronavirus infection lung, 204f

rat, 203 Parker's, 203

interstitial pneumonia, 204f Corynebacterium kutscheri, 201 epidermoid carcinoma lung, 124,

127 F344, 115t, 126t fibrosarcoma lung, 131t, 131t formaldehyde, nasoturbinate, 43f hemangiosarcoma lung, 131t Holtzman-SO, 115 interstitial pneumonia PVM, 208f lesions tail veins lung

Sprague-Dawley, 193t lung alveolar lipoproteinosis, 171f,

172f aspergillosis, 224f

brush cell, bronchiole, 94f Clara cell, bronchiole, 94f Pneumocystis carinii, 220f pneumonia virus mice, 207f squamous cell carcinoma, 124 tumor,131t

lymphosarcoma lung, 131t mesothelioma lung, 131t mouse hamster man, comparison squamous cell carcinoma, 132t

mucormycosis lung, 226f murine respiratory mycoplasmosis, 213f,214f

lung, 213 mycoplasmosis upper respira-tory tract, 78

Mycoplasma pUlmonis, 201 mycoplasmosis lung, 215f nasal cavity, 3f naturally occurring squamous cell

carcinoma lung, 126t neoplasms mucosa ethmoid turbinates, 47

normal bronchial lymphoid tissue, 215f

lung, 214f olfactory region F344, 53f Oregon, 115t Osborne-Mendel, 115t, 126t pneumocystosis lung, 218, 218f pneumonia virus mice infection lung

mouse, 206 polypoid adenoma maxilloturbinate,43f

nasal mucosa, 41 nose,42f

nasaladenoma,44f pulmonary lipidosis, 169t, 169, 170f radiation-induced squamous cell

carcinoma lung, 127 rat coronavirus infection lung, 203 reticulosarcoma lung, 131t sendai virus infection lung mouse,

195 serum biochemistry hypophysec

tomized, 169t Sherman, 115t sialodacryoadenitis virus infection

upper respiratory tr, 84 Sprague-Dawley, 115t

Crl COPS (SO), 115t HAP (SO), 115t

squamous cell carcinoma lung, 124, 125f,128f

F344, 129f nasal cavity, 58f

mucosa, 54, 59t nasoturbinate, 57f

Streptococcus pneumoniae, 201 subcutaneous mass, 55f submaxillary gland virus infection,

84,210 thrombus caudal vein male, 190f trachea, epithelium, 89f

normal,81f

seromucous glands, 91f type II alveolar cell, 96f ventral turbinate, 85f Wistar, 115t, 126t

Rats, chemically induced lung tumors F344,101t naturally occurring bronchiolar alveolar tumors, 115t

Renal adenocarcinoma, lung mouse metastasis, 145f transitional cell carcinoma, lung

mouse metastasis, 145f Reparative phase, sendai virus infection, 197f

Resolution phase, sendai virus infection, 200f

Respiratory cilium, human, 92f disease, murine chronic, 78, 213 epithelia, 6f epithelium, 6f, 7f, 8f, 9f, 36

syrian golden hamster, carcinogens, 33

mycoplasmosis, bronchiole murine, 216f bronchus murine, 216f chronic murine, 81 f lung rat, murine, 213 murine,79f rat, murine, 213f, 214f

mycoplasmosis upper respiratory tract rat, murine, 78

syncytial virus, RSV, 209 system, upper, 1 tract rat, murine respiratory mycoplasmosis upper, 78

sialodacryoadenitis virus infection up, 84

syrian hamster, squamous cell carcinoma upp, 62

tumor, mucoepidermoid, 39f Reticulosarcoma lung rat, 131t RIIl/Dm/Se mouse strain, 148t

mouse metastatic mammary tumor lung, 140f

strain, 148t RSV, respiratory syncytial virus, 209

Sarcocystis, 230 Sarcoma, lung mouse metastasis sub

cutaneous, 141f Schwannoma, lung mouse metastasis

malignant, 146f thrombi malignant, 146f

SDAV infection, 84, 210 sialodacryoadenitis virus, 210

Secondary tumors lung, 138 Secretory cell, 26f Segment, pigmented hair, 187f Sendai virus infection, 196f, 197f

lung mouse rat, 195 reparative phase, 197f resolution phase, 200f

Septal mucosa, normal nasal, 79f olfactory organ, 4f

Septum hamster lung, interalveolar, 96f

Seromucous glands rat trachea, 91f Serous cell rat bronchial epithelium,

90f Serum biochemistry

hypophysectomized rat, 169t Sherman, rat, 115t Sialodacryoadenitis, 84f, 85f

viral pneumonia, 211f virus, 86

infection lung mouse, 210 upper respiratory tract, 84

SDAV,210 Simple metaplasia, 35 Skin emboli lung, 186

metastasis lung, 150t particle, lung giant cell granuloma,

193t thromboarteritis, 193t

particles, lung, 188t Spironucleus, mouse, 201 Spleen, metastasis lung, 150t Spp, Leishmania, 227 Sprague-Dawley Cd/COPS/SD, rat,

115t HAP/SD, rat, 115t rat, 115t

lesions tail veins lung, 193t Squamous cell carcinoma, 47, 63f, 64f,

65f anterior nasal cavity, 56f lung F344 rat, 129f

hamster, 119f, 132t rat, 124, 125f, 128f

naturally occurring, 126t radiation-induced, 127

syrian hamster, 117 nasal cavity rat, 58f

mucosa,55f rat, 54, 59t

nasoturbinate rat, 57f pulmonary vein C57Bl J mouse,

130f rat lung, 124

mouse hamster man, comparison, 132t

upper respiratory tract syrian hamster, 62

papilloma, 33 tumor, 62, 117

cells, malignant, 64f, 65f, 118f epithelium, dysplastic, 63f metaplasia, 29f, 30f, 34, 36

alveolar, 199f lung hamster, 118f

Stage, predifferentiation, 11 Staphylococci, 221 Strain, A mouse, 148t, 148t

AlBrA mouse, 148t B6C3HFl mouse, 148t BALB/C/C3H/He mouse, 148t

cfC3H/Cb/Se mouse, 148t cfC3H mouse, 148t cfRIII mouse, 148t cNIV mouse, 148t

C3H/Cb/Se mouse, 148t

Subject Index 239

mouse, 148t C57BLl6J mouse, 148t CBA mouse, 148t Cf-l mouse, 148t DBA mouse, 148t OR mouse, 148t NIH white mouse, 148t RIII/Dm/Se mouse, 148t

mouse, 148t Streptobacillus moniliformis, 215, 221 Streptococcus pneumoniae, 215

rat, 201 Structure function lung, 89 Subcutaneous mass, rat, 55f

sarcoma, lung mouse metastasis, 141f

Submaxillary gland virus infection, rat, 84,210

Sulfonic acid, HEPES Hydroxyethyl piperazineethane, 28t

Sulfoxide, DMSO Dimethyl, 28t Swiss, mice, 106t Syncytia, bronchiole multiple

epithelial, 196f Syncytial virus RSV, respiratory, 209 Syrian golden hamster, carcinogens respiratory epithelium, 33

tracheal epithelium, 11 hamsters, explant cultures fetal trachea, 27

hamster, clear cell carcinoma larynx, 75

pleural mesothelioma, 133 radioactive materials, lung tumor,

132t squamous cell carcinoma lung,

117 upper respiratory tra, 62

System, upper respiratory, 1

Tachyzoites, toxoplasma, 227 Tail veins bleeding, 188t, 193t

hair thromboarteritis, 188t vessel, 193t

lung, lesions, 188t Sprague-Dawley rat, lesions,

193t periphlebitis, 188t, 193t phlebitis, 188t thrombophlebitis hair, 193t

Terreus pleura rat, Aspergillus, 225f Thrombi malignant schwan noma, lung

mouse, 146f Thromboarteritis hair particle, lung,

193t lung, 188t, 193t skin particle, lung, 193t tail veins hair, 188t

Thrombophlebitis hair, tail veins, 193t Thrombotic pulmonary artery, 187f Thrombus caudal vein male rat, 190f Tissue rat, normal bronchial lymphoid,

215f Torulopsis glabrata, Candida, 219,

222f

240 Subject Index

Toxicity bleomycin baboon, pulmonary, 165t

dog, pulmonary, 165t hamster, pulmonary, 165t mouse, pulmonary, 165t pheasant, pulmonary, 165t

Toxoplasma gondii, 227, 229 mouse, 201 tachyzoites, 227

Toxoplasmic pneumonia hamster, 228f nude mouse, 228f

Toxoplasmosis lung mouse hamster, 227

transmission, 229 Trachea, 11f

dysplasia, 35f epithelium rat, 89f explant culture fetal, 28

fetal,30f fetal,30f hamster, 22f

papillary mucoepidermoid tumor, 33f

hyperplasia, 35f normal rat, 81f papillary tumor, 37f seromucous glands rat, 91f syrian golden hamsters, explant

cultures fetal, 27 Tracheal epithelial cell Mycoplasma

pulmonis, 82f epithelium, 13f, 15f, 21f, 24f

hamster, 11f syrian golden hamster, 11

explant, fetal, 29f mucosa,81f

Tract rat, murine respiratory mycoplasmosis upper respiratory, 78

sialodacryoadenitis virus infection upper respirat, 84

Transitional cell carcinoma, lung mouse metastasis renal, 145f

Transmission toxoplasmosis, 229 Trimethoxycinnamaldehyde, 59t Trimethylaniline, 101 t Trophozoites, pneumocystis, 222f Trypanosoma cruzi, 227 Tumor emboli pulmonary artery, lung

mouse mammary, 141f epidermoid papillary, 33 lung mouse metastasis ovarian

granulosa cell, 144f RIll mouse metastatic mammary, 140f

mammary,138f mice radioactive materials, lung,

131t

muco-epidermoid papillary, 36f mucoepidermoid papillary, 33

respiratory, 39f olfactory region, 51f polypoid, 33 rat, lung, 131t squamous cell, 62, 117 syrian hamster radioactive materials,

lung,132t trachea hamster, papillary

mucoepidermoid, 33f papillary, 37f

Tumors, differential diagnosis papillary, 39

F344 rats, chemically induced lung, 10tt

lung mouse, metastatic, 138 secondary, 138

mice, frequency pulmonary, 106t pulmonary metastasis induced, 149t

papillary, 34 rats, naturally occurring bronchiolar alveolar, 115t

ultrastructure, papillary, 37 Turbinate, ethmoid, 4f

rat, ventral, 85f Turbinates rat, neoplasms mucosa

ethmoid,47 Tympanic bulla purulent exudate, 80f Type B, lung mouse metastatic

mammary adenocarcinoma, 143f I cell, 14f, 24f, 25f

mucous cells, 22f II adenoma, 103f

cell, 104f lung mouse, 102f, 103f, 104f

alveolar cell rat, 96f cell, 14f, 19f

adenoma, 102 lung mouse, 105f

alveolar, 102 ciliated cell, 16f, 18f epithelial cell, lung mouse, 163f

Ultrastructure, papillary tumors, 37 Untreated mice, pulmonary metastasis, 148t

Upper respiratory system, 1 tract rat, murine respiratory

mycoplasmosis, 78 sialodacryoadenitis virus

infect, 84 syrian hamster, squamous cell

carcino,62 Urinary bladder, metastasis lung, 150t

Vascular lesion bleomycin, lung mouse, 161f system, hair fragment emboli

pulmonary, 186 Vein male rat, thrombus caudal, 190f Veins bleeding, tail, 188t, 193t

hair thromboarteritis, tail, 188t vessel, tail, 193t

lung, lesions tail, 188t Sprague-Dawley, rat, lesions tail,

193t periphlebitis, tail, 188t, 193t phlebitis, tail, 188t thrombophlebitis hair, tail, 193t

VEN N-nitrosovinylethylamine, 28t Ventral turbinate rat, 85f Vessel, tail veins hair, 193t Vessels bleomycin, lung mouse

pulmonary blood, 162f Viral pneumonia, sialodacryo

adenitis, 211 f Virus infection JHV, Japanese

hemagglutinating, 195 lung mouse rat, sendai, 195

sialodacryoadenitis, 210 mouse HVM, hemagglutinating, 195

parainfluenza, 195 rat submaxillary gland, 84, 210 reparative phase, sendai, 197f resolution phase, sendai, 200f sendai, 196f, 197f upper respiratory tract rat,

sialodacryoadeni, 84 Japan HVJ, hemagglutinating, 195 mice infection lung mouse rat,

pneumonia, 206 pneumonia, 209 PVM, pneumonia, 206 rat lung, pneumonia, 207f

mouse K, 201 pneumonia, 201,206

parainfluenza, 202 pneumonia, 206 RSV, respiratory syncytial, 209 SDA V, sialodacryoadenitis, 210 sialodacryoadenitis, 86

Vomeronasal organ, 4f

White mouse strain, NIH, 148t Wistar, rat, 115t, 126t

Xylidine, 46t

Yeasts, fungal, 222f

Zygomycosis, 224

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Respiratory System